Bioinformatics

BIOINFORMATICS
TRENDS AND
METHODOLOGIES

Edited by Mahmood A. Mahdavi

Bioinformatics Trends and Methodologies
Edited by Mahmood A. Mahdavi

Published by InTech
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First published October, 2011
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Bioinformatics Trends and Methodologies, Edited by Mahmood A. Mahdavi
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Contents

Preface XI
Part 1 Bioinformatics in Biology 1
Chapter 1 Concepts, Historical Milestones and
the Central Place of Bioinformatics in
Modern Biology: A European Perspective 3
T.K. Attwood, A. Gisel, N-E. Eriksson and E. Bongcam-Rudloff
Part 2 Data Integration 39
Chapter 2 Data Integration in Bioinformatics:
Current Efforts and Challenges 41
Zhang Zhang, Vladimir B. Bajic, Jun Yu,
Kei-Hoi Cheung and Jeffrey P. Townsend
Chapter 3 Semantic Data Integration on
Biomedical Data Using Semantic Web Technologies 57
Roland Kienast and Christian Baumgartner
Part 3 Data Mining and Applications 83
Chapter 4 Vector Space Information Retrieval
Techniques for Bioinformatics Data Mining 85
Eric Sakk and Iyanuoluwa E. Odebode
Chapter 5 Massively Parallelized DNA Motif Search on FPGA 107
Yasmeen Farouk, Tarek ElDeeb and Hossam Faheem
Chapter 6 A Pattern Search Method for Discovering
Conserved Motifs in Bioactive Peptide Families 121
Feng Liu, Liliane Schoofs, Geert Baggerman,
Geert Wets and Marleen Lindemans
Chapter 7 Database Mining: Defining the Pathogenesis
of Inflammatory and Immunological Diseases 143
Fan Yang, Irene Hwa Yang, Hong Wang and Xiao-Feng Yang
VI Contents

Chapter 8 Data Mining Pubmed Identifies Core
Signalings and miRNA Regulatory Module in Glioma 157
Chunsheng Kang, Junxia Zhang, Yingyi Wang,
Ning Liu, Jilong Liu, Huazong Zeng, Tao Jiang,
Yongping You and Peiyu Pu
Part 4 Sequence Analysis and Evolution 171
Chapter 9 Significance Score of Motifs in Biological Sequences 173
Grgory Nuel
Chapter 10 A Systematic and Thorough Search
for Domains of the Scavenger Receptor
Cysteine-Rich Group-B Family in the Human Genome 195
Alexandre M. Carmo and Vattipally B. Sreenu
Chapter 11 Assessing Multiple Sequence
Alignments Using Visual Tools 211
Catherine L. Anderson, Cory L. Strope and Etsuko N. Moriyama
Chapter 12 Optimal Sequence Alignment
and Its Relationship with Phylogeny 243
Atoosa Ghahremani and Mahmood A. Mahdavi
Chapter 13 Predicting Virus Evolution 269
Tom Burr
Part 5 Protein Structure Analysis 287
Chapter 14 A Bioinformatical Approach to
Study the Endosomal Sorting Complex
Required for Transport (ESCRT) Machinery in
Protozoan Parasites: The Entamoeba histolytica Case 289
Israel Lpez-Reyes, Cecilia Bauelos,
Abigail Betanzos and Esther Orozco
Chapter 15 Structural Bioinformatics
Analysis of Acid Alpha-Glucosidase
Mutants with Pharmacological Chaperones 313
Sheau Ling Ho
Chapter 16 Bioinformatics Domain Structure Prediction and
Homology Modeling of Human Ryanodine Receptor 2 325
V. Bauerov-Hlinkov, J. Bauer, E. Hostinov, J. Gaperk,
K. Beck, . Borko, A. Faltnov, A. Zahradnkov and J. evk
Chapter 17 Identifying Enzyme Knockout
Strategies on Multiple Enzyme Associations 353
Bin Song, I. Esra Byktahtakn,
Nirmalya Bandyopadhyay, Sanjay Ranka and Tamer Kahveci
Contents VII

Part 6 Genome Analysis 371
Chapter 18 Using Bacterial Artificial Chromosomes to Refine
Genome Assemblies and to Build Virtual Genomes 373
Abhirami Ratnakumar, Wesley Barris,

Sean McWilliam and Brian P. Dalrymple
Chapter 19 Basidiomycetes Telomeres A Bioinformatics Approach 393
Luca Ramrez, Gmer Prez, Ral Castanera,
Francisco Santoyo and Antonio G. Pisabarro
Chapter 20 SNPpattern: A Genetic Tool to Derive
Haplotype Blocks and Measure Genomic
Diversity in Populations Using SNP Genotypes 425
Stephen J. Goodswen and Haja N. Kadarmideen
Chapter 21 Algorithms for CpG Islands Search:
New Advantages and Old Problems 449
Yulia A. Medvedeva
Chapter 22 Translational Oncogenomics and Human
Cancer Interactomics: Advanced Techniques
and Complex System Dynamic Approaches 473
I. C. Baianu
Part 7 Transcriptional Analysis 511
Chapter 23 In-silico Approaches for RNAi
Post-Transcriptional Gene Regulation:
Optimizing siRNA Design and Selection 513
Mahmoud ElHefnawi and Mohamed Mysara
Chapter 24 MicroRNA Targeting in Heart: A Theoretical Analysis 539
Zhiguo Wang
Chapter 25 Genome-Wide Identification of
Estrogen Receptor Alpha Regulated
miRNAs Using Transcription Factor Binding Data 559
Jianzhen Xu, Xi Zhou and Chi-Wai Wong
Part 8 Gene Expression and Systems Biology 575
Chapter 26 Quantification of Gene Expression
Based on Microarray Experiment 577
Samane F. Farsani and Mahmood A. Mahdavi
Chapter 27 On-Chip Living-Cell Microarrays for Network Biology 609
Ronnie Willaert and Hichem Sahli
VIII Contents

Chapter 28 Novel Machine Learning Techniques
for Micro-Array Data Classification 631
Neamat El Gayar, Eman Ahmed and Iman El Azab
Part 9 Next Generation Sequencing 653
Chapter 29 Deep Sequencing Data Analysis:
Challenges and Solutions 655
Ofer Isakov and Noam Shomron
Chapter 30 Whole Genome Annotation: In Silico Analysis 679
Vasco Azevedo, Vinicius Abreu, Sintia Almeida,
Anderson Santos, Siomar Soares, Amjad Ali, Anne Pinto,
Aryane Magalhes, Eudes Barbosa, Rommel Ramos,
Louise Cerdeira, Adriana Carneiro, Paula Schneider,
Artur Silva and Anderson Miyoshi
Part 10 Drug Design 705
Chapter 31 Designing of Anti-Cancer Drug Targeted to
Bcl-2 Associated Athanogene (BAG1) Protein 707
Amit Kumar, Kriti Verma and Amita Sinha

Preface

Bioinformatics is a growing multidisciplinary field of science comprising biology,
computer science, and mathematics. It is the theoretical and computational arm of
modern biology. In other words, bioinformatics is a tool in the hands of biologists for
analyzing huge amount of biological data available on mainstream public databases.
Currently, bioinformatics has gained variety of applications in agriculture, medicine,
engineering, and natural science. This book discusses a small portion of these
applications along with basic concepts and fundamental techniques in bioinformatics.
The first section is a review of history of bioinformatics and the pace of its
development in modern biology specifically in Europe. Section 2 and section 3 focus
on fundamental principles of data integration and data mining as basic skills in
bioinformatics. Data integration is now perceived a requirement in biology as the
volume of biological data continues to grow. Section 2 provides an overview on
integration of biomedical data using semantic web technologies and current efforts
and challenges. Data mining is another basic tool to search databases for conserved
regions, motifs, and regulatory modules effective in variety of diseases. Section 3
discusses these applications and basic approaches in data mining such as vector space
information. Section 4 concentrates on another aspect of bioinformatics, sequence
analysis. Sequences are analyzed to search for distribution of motifs, and search for
domains. Basic tool for this analysis is sequence alignment which is discussed in this
section in detail. Section 5 contains chapters on identification of specific structures in
proteins such as endosomal sorting complex, chaperons, and human receptors. These
structures are involved in different metabolic activities within the cell. Section 6 covers
those chapters that discuss role of bioinformatics in genomic studies. Some
applications of computational techniques in analysis of genomes such as SNP patterns,
CPG islands, and virtual genomes have been described in this section. Section 7
focuses on regulatory machinery and the role micro RNAs in this system. Micro RNAs
have recently been found to be important in regulatory networks. Some applications
have been discussed in chapters within this section. Gene expression and system level
understanding of expression process is one of the most interesting topics in
bioinformatics. Section 8 contains fundamental principles of identification of
differentially expressed genes from microarray data. The chapters in this section are
suitable for those who seek basic information on gene expression and integration of
this information into biological systems. Section 9 contains more advanced topics in
XII Preface

bioinformatics including next generation sequencing. In this section the authors
discuss more recent advances and technologies utilized in deep sequencing. The last
section describes one of the growing practical applications of bioinformatics i.e. drug
design. The ultimate goal of all theoretical analysis of biological data ought to be a
product that improves lives of human. This section discusses one of thousands of
efforts in designing a new drug for cancer treatment by means of bioinformatics.
Therefore, this book targets two types of readers: those who are new to bioinformatics
and are interested in basic methods and fundamental principles and those who seek
new approaches in bioinformatics. Both parties will benefit from studying this book.
In closing I wish to express my sincere sense of gratitude to all contributing authors,
publishing process manager, Petra Nenadic and publishing staff.

Mahmood A. Mahdavi
Ferdowsi University of Mashhad (FUM), Mashhad
Iran

Part 1
Bioinformatics in Biology

1
Concepts, Historical Milestones and the Central
Place of Bioinformatics in Modern Biology:
A European Perspective
T.K. Attwood
1
, A. Gisel
2
, N-E. Eriksson
3
and E. Bongcam-Rudloff
4

1
Faculty of Life Sciences & School of Computer Science, University of Manchester
2
Institute for Biomedical Technologies, CNR
3
Uppsala Biomedical Centre (BMC), University of Uppsala
4
Department of Animal Breeding and Genetics,
Swedish University of Agricultural Sciences
1
UK
2
Italy
3,4
Sweden
1. Introduction
The origins of bioinformatics, both as a term and as a discipline, are difficult to pinpoint.
The expression was used as early as 1977 by Dutch theoretical biologist Paulien Hogeweg
when she described her main field of research as bioinformatics, and established a
bioinformatics group at the University of Utrecht (Hogeweg, 1978; Hogeweg & Hesper,
1978). Nevertheless, the term had little traction in the community for at least another decade.
In Europe, the turning point seems to have been circa 1990, with the planning of the
Bioinformatics in the 90s conference, which was held in Maastricht in 1991. At this time, the
National Center for Biotechnology Information (NCBI) had been newly established in the
United States of America (USA) (Benson et al., 1990). Despite this, there was still a sense that
the nation lacked a long-term biology informatics strategy, particularly regarding
postdoctoral interdisciplinary training in computer science and molecular biology (Smith,
1990). Interestingly, Smith spoke here of biology informatics, not bioinformatics; and the
NCBI was a center for biotechnology information, not a bioinformatics centre.
The discipline itself ultimately grew organically from the needs of researchers to access and
analyse (primarily biomedical) data, which appeared to be accumulating at alarming rates
simultaneously in different parts of the world. The rapid collection of data was a direct
consequence of a series of enormous technological leaps that yielded what was considered,
at the time, unprecedented quantities of biological sequence information. Hot on the heels of
these developments was the concomitant wide-scale blossoming of algorithms and
computational resources necessary to analyse, manipulate and store these growing
quantities of data. Together, these advances gave birth to the field we now refer to as
bioinformatics.
When we look back, its clear that certain concepts and historical milestones were crucial to
the evolution of this new field. Those we think most important, and consequently


4
remember, depend largely on the perspective from which we view the emerging
bioinformatics landscape. This chapter takes a largely European standpoint, while
recognising that the development of bioinformatics in Europe was intimately coupled with
parallel advances elsewhere in the world, and especially in the USA. The history is intricate.
Here, we endeavour to recount the story as it unfolded along a number of tightly
interwoven paths, including the rise and spread of some of the technological developments
that spawned the data deluge and facilitated its world-wide propagation; of some of the
databases that developed in order to store the rapidly accumulating data; and of some of the
organisations and infrastructural initiatives that emerged to try to put some of those pivotal
databases on a more solid financial footing.
2. The seeds of bioinformatics
It is hard to pinpoint where and when the seeds of bioinformatics were originally sown.
Does the story start with Franklin and Goslings foundational work towards the elucidation
of the structure of DNA (Franklin & Gosling, 1953a, b, c), or with the opportunistic
interpretation of their data by Watson and Crick (Watson & Crick, 1953)? Do we fast-
forward to the ground-breaking work of Kendrew et al. (1958) and of Muirhead & Perutz
(1963) in determining the first three-dimensional (3D) structures of proteins? Or do we step
back, and focus on the painstaking work of Sanger, who, in 1955, determined the amino acid
sequence of the first peptide hormone? Or again, do we jump ahead to the progenitors of the
first databases of macromolecular structures and sequences in the mid-1960s and early 70s?
This era clearly heralded some of the most significant advances in molecular biology, as
witnessed by a string of Nobel Prizes at the time: e.g., Sangers Prize in Chemistry in 1958;
Watson, Crick and Wilkins shared Prize in Physiology or Medicine in 1962, following
Franklins death; and Perutz and Kendrews Prize in Chemistry, also in 1962. Clearly, in its
own way, each of these advances played an important part in the emergence of the vibrant
new field that we recognise today as bioinformatics.
As a humbling reference point, we have chosen to begin our story in the mid 1940s, with
Fred Sangers pioneering work on insulin. Sanger used a range of chemical and enzymatic
techniques to elucidate, for the first time, the order of amino acids in the primary structure
of a protein. Back then, this was a tremendously complex puzzle to tackle, and its
completion required the successful resolution of many different challenges over several
years. That this was a difficult incremental process is illustrated by the fact that, between
1945 and 1955, each step was published in a separate, stand-alone article. All in all,
something like 10 papers detail the series of experiments that led to the eventual
determination of the sequences of bovine insulin (e.g., Sanger, 1945; Sanger & Tuppy, 1951a,
b; Sanger & Thompson, 1953a,b; Sanger et al., 1955; Ryle et al., 1955) and of ovine and
porcine insulins (Brown et al., 1955). This was ground-breaking work, and had taken 10
years to complete. Incredibly, the 3D structure would not be known for another 14 years
(Adams et al., 1969). The primary and tertiary structures of this historical protein are
illustrated in Figure 1.
Such was the enormity of manual sequencing projects that it was many years before the
sequence of the first enzyme (ribonuclease) was determined. Work on this protein began in
1955. After preliminary studies in 1957 and 1958, the first full draft sequence was published
in 1960 (Hirs et al., 1960). During the months that followed, the draft was meticulously
refined, and a final version was published 3 years later (Smyth et al., 1963). Crucially, this 8-
Concepts, Historical Milestones and
the Central Place of Bioinformatics in Modern Biology: A European Perspective

5
year project paved the way for the elucidation of the proteins 3D structure indeed,
without the sequence information, the electron density maps could not have been
meaningfully interpreted (Wyckoff et al., 1967). Knowledge of the primary structure of this
small protein thus provided a vital piece of a 3D jigsaw puzzle that was to take a further 4

Fig. 1. Illustration of a) the primary structure of bovine insulin, showing intra- and
interchain disulphide bonds connecting the a and b chains; and b) its zinc-coordinated
tertiary structure (2INS), revealing two molecules in the asymmetric unit, and a hexameric
biological assembly.
years to solve. Viewed in the light of the high-throughput sequence and structure
determinations of today, these prolonged time-scales now seem almost inconceivable.
Notwithstanding the challenges, however, the potential of peptide sequencing technology to
aid our understanding of the biochemical functions and evolutionary histories of particular
proteins, and to facilitate their structural analysis, was compelling. Consequently, the
sequences of many other proteins were soon deduced. In the early 60s, amongst the first to
appreciate the value of biological sequences, and particularly the ability to deduce
evolutionary relationships from them, was Margaret Dayhoff. To facilitate her research and
the work of others in the field, she began to collect all protein sequences then available,
ultimately publishing them in book form this was the first Atlas of Protein Sequence and
Structure (Dayhoff et al., 1965), often simply referred to as the Atlas. It may seem amusing to
us now, but in a letter she wrote in 1967, she observed, There is a tremendous amount of
information regarding the evolutionary history and biochemical function implicit in each sequence
and the number of known sequences is growing explosively [our emphasis]. We feel it is
important to collect this significant information, correlate it into a unified whole and interpret it
(Dayhoff, 1967; Strasser, 2008). With the publication of the first Atlas, that explosive growth
amounted to 65 sequences!


6
In the decade that followed, time-consuming manual processes were gradually superseded
with the advent of automated peptide sequencers, which increased the rate of sequence
determination considerably. Meanwhile, another revolution was taking place, heralded by
the elucidation of the 3D structures of the first proteins, those of myoglobin and
haemoglobin, respectively (Kendrew et al., 1958; Muirhead and Perutz, 1963). Building on
the ongoing sequencing work, this advance set the scene for an exciting new era in which
structure determination took centre stage in our quest to understand the biophysical
mechanisms that underpin biochemical and evolutionary processes. In fact, so seductive
was this approach that many more structural studies were initiated, and the numbers of
deduced protein structures grew accordingly.
3. The development and spread of databases, organisations and
infrastructures
Key to handling this burgeoning information was the recruitment of computers to help
systematically analyse and store the accumulating sequence and structure data. At this time,
the idea that molecular information could be collected within, and distributed from,
electronic repositories was not only very new but also posed significant challenges. Just
consider, for a moment, that concepts we take for granted today (email, the Internet, the
World Wide Web) had not yet emerged; there was therefore no easy way to distribute data
from a central database, other than by posting computer tapes and disks to individual users,
at their request. This model of data distribution was clearly rather cumbersome and slow; it
was also relatively costly, and led some of the first database pioneers to adopt pricing
and/or data-sharing policies that threatened to drive away many of their potential users.
3.1 The Protein Data Bank (PDB)
One of the earliest, and hence now oldest, of scientific databases was established in 1965 at
the Cambridge Crystallographic Data Centre (CCDC), under the direction of Olga Kennard
(Kennard et al., 1972; Allen et al., 1991) this was a repository of small-molecule crystal
structures termed the Cambridge Structural Database, or CSD. The CSD, which originated
as a traditional printed dissemination, ultimately assumed an electronic form so that
Kennard could fulfill a dream, which she shared with J.D.Bernal, to be able to use data
collections to discover new knowledge, above and beyond the results yielded by individual
experiments (Kennard, 1997).
In 1971, a few years after the creation of the CSD, at a Cold Spring Harbor Symposium on
the Structure and Function of Proteins at the Three Dimensional Level, Walter Hamilton and
colleagues discussed the possibility of creating a similar kind of bank for protein
coordinate data. Key to their proposal was that this archive should be mirrored at sites in
the UK and the USA (Berman, 2008). Consequently, Hamilton volunteered to set up the
master copy of the American bank at the Brookhaven National Laboratory (BNL), while
Kennard subsequently agreed to host the European copy and to extend the CCDC small
molecule format to accommodate protein structural data (Kennard et al., 1972; Meyer, 1997).
Thus was born the Protein Data Bank (PDB); this was to be operated jointly by the CCDC
and BNL, and where possible, distributed on magnetic tape in machine-readable form.
News of its establishment was announced in a short bulletin in October that year (Protein
Data Bank, 1971); its first release held 7 structures (Berman et al., 2000). Interestingly,
Kennard viewed the PDB as a prototype for the EMBL data library, which was to materialise
a decade later (Smith, 1990).

7
By 1973, the PDB was fully operational (Protein Data Bank, 1973). In August that year, the
body of data it had been established to store amounted to 9 structures (see Table 1). Kennard
and co-workers knew that the success of the resource was ultimately dependent on the
support of the crystallography community in providing their data; but gaining sufficient
community momentum to back the initiative was clearly a long, drawn-out process: note,
for example, that the structure of ribonuclease, which had been determined 6 years earlier,
was not yet listed amongst its holdings.

Protein structures
1 Cyanide methaemoglobin V from sea lamprey
2 Cytochrome b
5

3 Basic pancreatic trypsin inhibitor
4 Subtilisin BPN (Novo)
5 Tosyl -chymotrypsin
6 Bovine carboxypeptidase A
7 L-Lactate dehydrogenase
8 Myoglobin
9 Rubredoxin
Table 1. PDB holdings, August 1973.
Over the next 4 years, the number of structures acquired by the PDB grew slowly. By 1977,
the archive also included the structure of a transfer RNA (tRNA), and hence the name
Protein Data Bank was thought something of a misnomer (Bernstein et al., 1977).
Nevertheless, despite this reservation, the name stuck, and the resource (which today
includes more than 5,000 nucleic acid and protein-nucleic acid complexes) is still referred to
as the PDB. Interestingly, at that time, the database contained 77 sets of atomic coordinates
relating to 47 macromolecules, highlighting a significant level of redundancy. Coupled with
their ongoing concerns about the pace of growth of the archive, perhaps this explains why
the Berstein et al. paper was published verbatim in May and November of 1977, and again in
January 1978, in three different journals (Bernstein et al., 1977a, b; 1978)? Whatever the real
reasons, growth of the PDB compared to the CSD (~6,000 vs. ~150,000 structures in 1996)
was slow (Kennard, 1997), and the number of unique structures remained relatively small
by 1992, the level of redundancy in the resource had been calculated to be ~7-fold (Berman,
2008; Hobohm et al., 1992).
In 1996, shortly after the establishment of the European Bioinformatics Institute (EBI) near
Cambridge, UK, a new database of macromolecular structures was created this was the E-
MSD (Boutselakis et al., 2003). Building directly on PDB data, E-MSD was originally
conceived as a pilot study to explore the feasibility of exploiting relational database
technologies to manage structural data more effectively. In the end, the pilot project led to
the creation of a database that was successful in its own right, and the E-MSD thereby
became established as a major EBI resource.
During this period, a concerted effort was made to hasten the pace of knowledge acquisition
from structural studies. Part of the motivation was to build on the still-limited number of
structures available in the PDB, and partly also to address its growing level of redundancy.
The idea was to establish a program of high-throughput X-ray crystallography the so-
called Structural Genomics Initiative (SGI) (Burley et al., 1999). Several feasibility studies had


8
already been launched and, in light of the broad-sweeping vision of the SGI, it had become
clear that coping with high-throughput structure-determination pipelines would require
new ways of gathering, storing, distributing and serving the data to end users. One of the
PDBs responses to this, and to the many challenges that lay ahead, was the formation of a
new management structure. This was to be embodied in a 3-membered Research
Collaboratory for Structural Bioinformatics (RCSB): the consortium included Rutgers, The
State University of New Jersey; the San Diego Supercomputer Center at the University of
California; and the Center for Advanced Research in Biotechnology of the National Institute
of Standards and Technology (Berman et al., 2000; Berman et al., 2003). Once the consortium
was established, the BNL PDB ceased operations and the RCSB formally took the helm on 1
July, 1999.
With the RCSB PDB in the USA, the E-MSD established in Europe, and a sister resource
(PDBj) subsequently announced in Japan (Nakamura et al., 2002), structure collection efforts
had clearly taken on an international dimension. In consequence, in 2003, the 3 repositories
were brought together beneath an umbrella organisation known as the worldwide Protein
Data Bank (wwPDB), to streamline their activities and maintain a single, global, publicly
available archive of macromolecular structural data (Berman et al., 2003). By 2009, perhaps
to align its nomenclature in a more obvious way with its consortium partners, E-MSD was
renamed PDBe (Velankar et al., 2009). Today, the RCSB remains the archive keeper, with
sole write-access to the PDB, controlling its contents, and distributing new PDB identifiers to
all deposition sites. In February 2011, the archive housed 71,415 structures.
3.2 The EMBL nucleotide sequence data library
Despite the advances in protein sequence- and structure-determination technologies
between the mid-1940s and -70s, sequencing nucleic acids had remained problematic. The
key issues related to size and ease of molecular purification. It had proved possible to
sequence tRNAs, largely because theyre short (typically less than 100 nucleotides long) and
individual molecules could, with some effort, be purified; but chromosomal DNA molecules
are in a different league, containing many millions of nucleotides. Even if such molecules
could be broken down into smaller chunks, purification was a major challenge. The longest
fragment that could then be sequenced in a single experiment was ~500bp; and yields of
potentially around half a million fragments per chromosome were simply beyond the
technology of the day to handle.
During the mid 70s, however, Sanger had developed a technology (to become known as the
Sanger method) that made it possible to work with much longer nucleotide fragments: this
allowed completion of the sequencing of the 5,386 bases of the single-stranded
bacteriophage X174 (Sanger et al., 1978), subsequently permitting rapid and accurate
sequencing of even longer sequences an achievement of sufficient magnitude to earn him
his second Nobel Prize in Chemistry, in 1980. With this technique, he went on to sequence
human mitochondrial DNA (Anderson et al., 1981) and bacteriophage (Sanger et al., 1982).
These were landmark achievements (see Table 2), providing the first direct evidence of the
phenomenon of overlapping gene sequences and of the non-universality of the genetic code
(Sanger, 1988; Dodson, 2005). But it was automation of these techniques from the mid-80s
that significantly increased productivity, and began to make the human genome a realistic
target.
Together, these advances prepared the way for a new revolution, one that would rock the
foundations of molecular biology and make the gathered fruits of all sequencing efforts

9
before it appear utterly inconsequential. Here, then, was a dramatic turning point: for the
first time, it dawned on scientists that the new sequencing machines were shunting the
bottlenecks away from data production per se and onto the requirements of data
management: the rate limiting step in the process of nucleic acid sequencing is now shifting from
data acquisition towards the organization and analysis of that data (Gingeras & Roberts, 1980).
This realisation had profound consequences in both Europe and the USA, as a centralised
data bank now seemed inescapable as a tool for managing nucleic acid sequence
information efficiently.

Year Protein RNA DNA No. of residues
1935 Insulin 1
1945 Insulin 2
1947 Gramicidin S 5
1949 Insulin 9
1955 Insulin 51
1960 Ribonuclease 120
1965 tRNA
Ala
75
1967 5S RNA 120
1968 Bacteriophage 12
1977 Bacteriophage X 174 5,375
1978 Bacteriophage X 174 5,386
1981 Mitochondria 16,569
1982 Bacteriophage 48,502
1984 Epstein-Barr virus 172,282
2004 Homo sapiens 2.85 billion
Table 2. Sequencing landmarks.
So, the race was on to establish the first nucleotide sequence database. First past the post, in
1980, was the European Molecular Biology Laboratory (EMBL) in Heidelberg, who set up
the EMBL data library. After an initial pilot period, the first release of 568 sequences was
made in June 1982. The aim of this new resource was not only to make nucleic acid sequence
data publicly available and encourage standardisation and free exchange of data, but also to
provide a European focus for computational and biological data services (Hamm &
Cameron, 1986).
From the outset, it was recognised that maintenance of such a centralised repository, and of
its attendant services, would require international collaboration. In the UK, a copy of the
EMBL library was being maintained at Cambridge University, together with its manual,
indices and associated sequence analysis, and search and retrieval software. This integrated
system also provided access to the library of sequences then being developed at Los Alamos,
GenBank (Kanehisa et al., 1984). It makes fascinating reading to learn that, this system is
presently being used by over 30 researchers in eight departments in the University and in local
research institutes. These users can keep in touch with each other via the MAIL command! With the
support of the Medical Research Council (MRC), the Cambridge services were extended to
the wider UK community on the Joint Academic network (JANET) (Kneale & Kennard,
1984). As with the PDB before it, it was important not only to push the data out to
researchers, but also to pull their data in. Hence, a further planned development was to


10
centralise collection of nucleic acid data from UK research groups, and to periodically
transfer the information to the EMBL library. It was hoped that this would minimise both
data-entry errors and the workload of EMBL staff at a time when the number of sequence
determinations was predicted to increase greatly (Kneale & Kennard, 1984). Of course, the
size of this great increase could hardly have been predicted; in December 2010, the
database contained 199,720,869 entries.
3.3 GenBank
The birth of GenBank, in December 1982, brought 606 sequences into the public domain. A
consensus had emerged on the necessity of creating an international nucleic acid sequence
repository at a scientific meeting at Rockefeller University in New York, in March 1979. At
that time, several groups had expressed a desire to be a part of this endeavour, including
those led by Dayhoff at the National Biomedical Research Foundation (NBRF); Walter Goad
at Los Alamos National Laboratories; Doug Brutlag at Stanford; Olga Kennard and Fred
Sanger at the MRC Laboratory in Cambridge; and Ken Murray and Hans Lehrach at the
EMBL (Smith, 1990), all of whom had begun to create their own nucleotide sequence
collections. However, it took the best part of 3 years for an appropriate funding model to
emerge from the US National Institutes of Health (NIH), by which time the EMBL data
library had already been publicly available for 6 months under the direction of Greg Hamm.
By then, 3 proposals remained on the table for NIH support: 2 of these were from Los
Alamos (one with Bolt, Beranek and Newman (BBN), the other with IntelliGenetics), and the
third from NBRF. To the surprise of many, the decision was made in June 1982 to establish
the new GenBank resource at Los Alamos (in collaboration with BBN, Inc.) rather than at the
NBRF (Smith, 1990; Strasser, 2008).
Although there was a general sense of relief that a decision had finally been made, some
members of the community (and doubtless Dayhoff herself) felt that the NBRF would have
been a more appropriate home for GenBank, particularly given Dayhoffs successful track
record as a curator of protein sequence data (Smith, 1990). Los Alamos, by contrast,
although undoubtedly offering excellent computer facilities, was probably best known for
its role in the creation of atomic weapons this was not an obvious environment in which to
establish the nations first public nucleotide sequence database. The crux of the matter
seemed to rest with the different philosophical approaches embodied in the NBRF and Los
Alamos proposals, particularly as they related to scientific priority, data sharing/privacy
and intellectual property policies. Dayhoff had intended to continue gathering sequences
directly from literature sources and from bench scientists, and wasnt interested in matters
of history or priority (Eck & Dayhoff, 1966); the Los Alamos team, on the other hand,
advocated the collaboration of journal editors in making the publication of articles
contingent on authors yielding their sequence data to the database. This latter approach was
particularly compelling, as it would allow scientists to assert priority, and to keep their
research results private until formally published and their provenance established; perhaps
more importantly, it was unencumbered by proprietary interest in the data. Unfortunately,
the fact that Dayhoff had prevented redistribution of NBRFs protein sequence library and
sought revenues from its sales (albeit only to cover costs) worked against her allowing the
data to become the private hunting grounds of any one group of researchers was considered
antithetical to the spirit of open access (Strasser, 2008). That the data and associated software
tools should be free and open was thus paramount; it is perhaps ironic, then, that the site
chosen for the database was within the secured area of what many in the community may
have darkly perceived as The Atomic City (en.wikipedia.org/wiki/The_Atomic_City).

11
As an aside, its interesting that the vision of free data and programs was advocated so
strongly at this time, not least because there was no funding model to support it! And
precisely the same arguments are still being vehemently propounded today with regard to
free databases, free software and free literature (e.g., Lathrop et al., 2011). But even now,
database funding remains an unsolved and controversial issue: as Olga Kennard put it
almost 15 years ago, Free access to validated and enhanced data worldwide is a beautiful dream.
The reality, however, is more complex (Kennard, 1997).
Returning to our theme, perhaps the final nail in the coffin of Dayhoffs proposal was that
the NBRF had only limited means of data distribution (via modems), whereas the Los
Alamos outfit had the enormous benefit of being able to distribute their data via ARPANET,
the computer network of the US Department of Defense. Together, these advantages were
sufficient to swing the pendulum in favour of the Los Alamos team.
But the new GenBank did not, indeed could not, function in isolation. From its inception, it
evolved in close collaboration with the EMBL data library and, from 1986 onwards, also
with the DNA Data Bank of Japan. Although the databases were not identical (each with its
own format, naming convention, and so on), the teams adopted common data-entry
standards and data-exchange protocols in order to improve data quality and to manage both
the growth of the resource and the annotation of its entries more effectively. Of this
collaborative process, Temple Smith commented in 1990, By working out a division of labor
with the EMBL and newer Japanese database efforts, and by involving the authors and journal
editors, GenBank and the EMBL databases are currently keeping pace with the literature. Today,
the boot seems to be very much on the other foot, as the literature can no longer keep up
with the data: by February 2011, GenBank contained 132,015,054 entries, presenting
insurmountable annotation hurdles! (Note that this appears smaller than the size of the
EMBL data library because GenBank doesnt report sequences from Whole Genome
Shotgun projects in its total). Perhaps not surprisingly, the initial funding for GenBank was
insufficient to adequately maintain this growing mass of data; hence, responsibility for its
maintenance, with increased funding under a new contract, passed to IntelliGenetics in
1987; then, in 1992, it became the responsibility of the NCBI, where it remains today (Benson
et al., 1993; Smith, 1990).
3.4 The PIR-PSD
To some extent, the gathering momentum of nucleic acid sequence-collection efforts had
begun to overshadow the steady progress being made in the world of protein sequences,
most notably with the Atlas. By October 1981, this had run into its fifth volume, a large book
with three supplements, listing more than 1,660 proteins. This information, as with all data
collections, required constant updating and revision in the light both of new knowledge and
of new data appearing in the literature. Moreover, as the community had become
increasingly keen to harness the efficiency gains of central data repositories, and more
databases were appearing on the horizon, making and maintaining cross-references to
database entries, of necessity, had to become part of data-annotation and update processes if
scientists were to be able to exploit new and existing sequence data fully. Under the
circumstances, continued publication of the Atlas in paper form simply became untenable:
the time was ripe to exploit the advances in computer technology that had given rise to the
CSD, the PDB, the EMBL data library and GenBank. In 1984, the Atlas was consequently
made available on computer tape as the Protein Sequence Database (PSD).


12
Later, in 1986, in order to facilitate protein sequence analysis more broadly, the NBRF
established the Protein Identification Resource (PIR) (George et al., 1986). This new online
system included the PSD, several bespoke query and analysis tools (e.g., the Protein
Sequence Query (PSQ), SEARCH and ALIGN programs), and a new, efficient search
program, FASTP. The latter was a modification of an earlier algorithm for searching protein
and nucleic acid sequences (Wilbur & Lipman, 1983). Interestingly, given that the number of
deduced sequences had, by that time, grown into the thousands, the great advantage of
Wilbur and Lipmans method was considered to be its speed. Indeed, their paper reported a
substantial reduction in the time required to search a data bank. Improving on this even further,
the new FASTP algorithm was able to compare a 200-amino-acid sequence to the 2,677
sequences of the PSD in less than 2 minutes on a minicomputer, and less than 10 minutes on a
microcomputer (IBM PC) (Lipman & Pearson, 1985). Looking back, such search times on
such small numbers of sequences seem incredibly slow; at the time (when a contemporary
algorithm required 8 hours for the same search), they were revolutionary.
As the PIR was built on NBRFs existing resources, it also made available its DNA databank
(Dayhoff et al., 1981a) and associated software tools, together with copies of GenBank and
the EMBL data library; it also retained the NBRFs cost-recovery model, levying a charge for
copies of its databases on magnetic tape and an annual subscription fee for use of its online
services in 1988, these amounted to $200 per tape release and $350 per annum respectively
(Dayhoff et al., 1981b; Sidman et al., 1988). By 1992, the PSD had shown steady growth, with
increasing contributions from European and Asian protein sequence centres most notably,
from MIPS (Martinsried, Germany) and from JIPID (Tokyo, Japan). Accordingly, a tripartite
collaboration was established, termed PIR-International, to formalise these relationships and
establish and disseminate a comprehensive set of protein sequences (Barker et al., 1992). By
this time, charging for access to the resource was no longer mentioned, possibly both as a
consequence of this more formal distribution arrangement and the advent of browsers like
Mosaic, which had suddenly and dramatically changed the way that information could be
broadcast and received over the World Wide Web (or, simply, the Web). In 1997 PIR
changed its name to the Protein Information Resource (George et al., 1997) and, by 2003,
with 283,000 sequences (Wu et al., 2003), the PSD was the most comprehensive protein
sequence database in the world.
3.5 Swiss-prot
While these events were taking place, a newly qualified Swiss student (who, as a teenager,
had been interested in space exploration and the search for extraterrestrial life) attempted to
embark on a Masters project involving both wet and dry work this was Amos Bairoch.
The experimental side of his project immediately hit problems when it was discovered that
the new mass spectrometer he was to have used didnt work properly. He therefore set to
work instead developing protein sequence analysis programs on the computer system
running the spectrometer. These were the first steps towards creating the software system
that was later to be known as PC/Gene, and was to become the most widely used PC-based
sequence analysis package of its day (Bairoch, 2000).
Part of what made this software suite unique was its focus on proteins at a time when the
analysis of nucleotide sequences was very much in vogue. In creating these tools, Bairoch
entered >1,000 protein sequences into his computer by hand: some of these he gleaned from

13
the literature; most were taken from the Atlas, which had not yet been released in electronic
form. Of course, this was an immensely tedious process, and was also highly error-prone.
Realising this, and anxious to avoid such problems for others in future, he wrote a letter to
the Biochemical Journal recommending that researchers publishing protein and peptide
sequences should compute checksums to facilitate the detection of typographical and keyboard
errors (Bairoch, 1982). As part of the letter, he illustrated the computation of such a
checking number for an imaginary peptide, as shown in Figure 2. Although this
recommendation was never widely adopted in publishing circles, Bairoch was at least able
to ensure that it was implemented in his own database.

Fig. 2. Computation of a checking number (CN) for an imaginary peptide, as published in a
letter to the Biochemical Journal in 1982. The journal editors either didnt notice, or chose to
ignore, the hidden message in the peptide. Reproduced with permission, from Bairoch, A.
(1982), Biochemical Journal, 203, 527-528. the Biochemical Society
Several other important developments were to emerge from the work of this enthusiastic
and industrious student. For the analysis software he was developing, he needed to
distribute both a nucleotide and a protein sequence database. In 1983, he acquired a
computer tape containing 811 sequences in version 2 of the EMBL data library; for his
protein sequence database, he initially used the sequences hed typed in for his Masters
project. However, the following year, he received the first electronic copy of the Atlas. He
was quick to appreciate the advantages and disadvantages of the PIR and EMBL formats,
recognising that converting the manually annotated data of the former into something like
the semi-structured format of the latter could produce a resource with the strengths of both
he called this PIR+ and released it side-by-side with his software package, PC/Gene,
which by that time hed commercialised through IntelliGenetics (Bairoch, 2000).
Use of the publicly available PIR data-set in this way was not without its problems.
Amongst other, deeper, issues were the difficulty of parsing PIR files to extract specific
information (e.g., relating to post-translational modifications (PTMs), etc.); the lack of
functional annotations for some of the newer entries; the lack of cross-referencing to the
parent DNA of a given protein sequence; and so on. Somewhat ironically, given what he
went on to achieve, Bairoch has written of this period, As I was not interested in building up
databases I kept sending letters to PIR to ask them to remedy this situation. But his pleas met
with little success. In the summer of 1986, in the face of increasing demand for
unencumbered access to his database, he decided to release PIR+ independently of
PC/Gene, to make it freely available to the entire research community. The new, public
version of the database was released on 21 July 1986 and contained ~3,900 sequences (the
exact number is unknown as the original floppy disks have been lost!) This new resource


14
was called Swiss-Prot (Bairoch & Boeckmann, 1991), and was to become the foremost
manually annotated database of protein sequences in the world.
3.6 The European Molecular Biology Network (EMBnet)
It is interesting that, during this era, the distribution of databases like the EMBL data library,
PIR, Swiss-Prot and so on, was still largely effected by the exchange of computer tapes and
disks. By this time, a variety of computer networks had begun to evolve: the first such
network, ARPANET (which began life with 4 nodes in late 1969), was the progenitor of the
Internet, and was superseded by it in 1983 recall, it was partly owing to the existence of
ARPANET that GenBank was established at Los Alamos. Other networks that offered
gateways into the Internet later merged with it, including Usenet and BITNET; commercial
and educational networks, such as Telenet (or Sprintnet), Tymnet, Compuserve and JANET,
were interconnected with it in the 1980s.
In 1988, Chris Sander at the EMBL helped to establish a new network, EMBnet, to
disseminate data, knowledge and services, to support and advance molecular biology and
biotechnology research across Europe. A major driver for creating EMBnet was the need for
local access to databases such as the EMBL data library from centralised sources. Essentially,
this is because scientists were now demanding to use client workstations with Graphical
User Interfaces (GUIs) that provided real-time interaction with their back-end data/analysis
servers. At the time, high-speed data communication across Europe was in its infancy, and
access to remote computers using ordinary command-line oriented terminals was too slow.
It was clear that communication delays could be eliminated if servers held copies of data
locally; the sheer amount of compute resources needed for European research in this field
also pointed to a distributed solution (note that computer cluster technology only gained
widespread acceptance much later). Thus, an organised way of distributing data and
resources from the EMBL to its member states had to be established.
The concept of a network of national nodes, each serving its country with up-to-date
biological databases and also providing compute resources for data analysis, was
formulated. It was given the name the European Molecular Biology network, EMBnet. The
first practical steps were taken in the spring of 1988 to solicit feedback from scientists
around Europe; and in July 1988, the first EMBnet Workshop was organised at EMBL, with
participants from EMBL, Daresbury (UK), CITI2 (France), the CAOS/CAMM Centre (the
Netherlands) and Hoffmann-La Roche. In November of that year, the EMBL Director
General corresponded with EMBL Council members, encouraging them to stimulate local
processes to identify regional EMBnet nodes. As more countries joined the network (France,
Sweden, the UK, the Netherlands, Spain, Israel, Norway, Italy and Denmark, with
Switzerland, West Germany, Austria, Greece and Finland waiting in the wings), EMBnet
received its first European grant under the BRIDGE framework, in 1991.
The principal project objective was to promote EMBnet as a computer network for European
bioinformatics. Service provision and knowledge sharing was to be orchestrated primarily
by 'National Nodes', with government mandates to support their local communities,
especially by providing access to bioinformatics data synchronised with the EMBL,
GenBank and DDBJ central data repositories in time, the network also attracted a number
of Specialist and Industrial Nodes, whose resources and know-how were seen to
complement those of its National Nodes (this arrangement of cooperating Nodes is
illustrated in Figure 3).
Most EMBnet Nodes had VAX computers, and the original intention was to use DECNET as
the underlying transport protocol. However, after a short, but expensive, period of using

15
X25/DataPak, this was replaced by a TCP/IP-package called MultiNet, which was licensed
for all EMBnet Nodes from SRI (Stanford Research Institute). FTP-transmissions of database
updates were often interrupted by network problems, and, to overcome the need for
frequent re-transmissions, the NDT (Network Data Transfer, later xNDT for extended NDT)
protocol was developed at the Swedish EMBnet Node at Uppsala Biomedical Centre, by
Peter Gad. It was given a so-called 'systems well-known port' (embl-ndt, 394/udp,# EMBL
Nucleic Data Transfer) by the Internet authorities, and is thus in good company with, for
example, Telnet (port 23) and FTP (ports 20, 21). For a few years, (x)NDT, and its
accompanying suite of client-server programs, was the method par preference, used at
almost all EMBnet Nodes to keep their local databases updated. NDT took care of the
transmission (database) entry by entry and didnt have to re-start following network
interruptions. The Greek node, situated in Crete, only had a modem connection to the
mainland, and benefited hugely from using the xNDT-suite. Indeed, at the time the
European Bioinformatics Institute was established (when the EMBL Data Library moved
from Heidelberg to Cambridge), most of the nucleotide sequence database update traffic in
Europe was routed via the Swedish node using xNDT.

Fig. 3. Illustration of the relationship between the different Nodes of the early EMBnet: some
National Nodes had either Specialist or Industrial Nodes affiliated with them; some had
both; some had neither. Today, 31 National and Specialist Nodes contribute to the Network.


16
By the early 90s, biomolecular databases could be accessed across the Internet by means of
the WAIS and Gopher network retrieval systems; and, under the auspices of EMBnet,
Reinhard Doetlz had developed a new network access protocol, HASSLE, the Hierarchical
Access System for Sequence Libraries in Europe (Doelz, 1994). But it was the advent of
graphical Web browsers (first, Mosaic from the National Center for Supercomputing
Applications in 1993, and then Netscape Navigator in 1994) that revolutionised the
processes of database dissemination and information consumption literally, at the click of
a mouse button.
Of course, browsers allowed data and documents of all kinds to be instantly shared, and
individuals and organisations across the globe were quick to establish their own unique
Web presence. EMBnet was no exception, and embraced the Web as a means of
communicating more effectively with its widening community, in particular by publishing a
regular newsletter, EMBnet.news. The newsletter was designed to provide reports and
updates on its internal and international activities and achievements, together with technical
and scientific papers on new developments in bioinformatics, computational biology and
biocomputing. In 2000, the organisation provided an educational grant to help support the
creation of the peer-reviewed journal Briefings in Bioinformatics (BiB) and, as a mark of its
own success, EMBnet.news is also now in the process of transitioning to a peer-reviewed
journal.
From the outset, EMBnet has promoted the development of distributed computing services
to share workload among international servers; it has contributed to the development and
maintenance of advanced database systems; it has been an advocate of the deployment of
Grid technologies for the life sciences through its contributions to major European Grid
projects; it developed, and continues to promote the use of, an e-learning system both to
support distance learning in bioinformatics and to complement face-to-face bioinformatics
teaching and training; and it is committed to bringing the latest software and algorithms to
users, free of charge.
The combined expertise of its Nodes has allowed EMBnet to provide services to its local
European life science communities with far greater effect than could be achieved by any of
its individual Nodes in isolation. Following this success, a variety of Nodes world-wide
have joined EMBnet such that, today, the network is global, with many countries from Asia,
Africa and America joining in recent years (including Sri Lanka, Pakistan, Kenya and Costa
Rica). Currently, the network connects 31 member Nodes extending over 27 countries;
together, the Nodes continue to work to disseminate data, to share compute resources and
to provide training support, reaching out to many thousands of users.
3.7 PROSITE
While EMBnet was being conceived, before the Internet had truly taken off, and while
bioinformatics was still in the throes of been born, the computer savvy molecular biologists
of the day were still busily swapping biomolecular databases on magnetic tapes and
computer disks. Perhaps an inevitable consequence of the systematic collection of protein
and nucleotide sequences in this way was the need to organise and classify these molecular
entities in meaningful ways. The first endeavour to categorise protein sequences into
evolutionarily related families, and to provide the diagnostic means to detect potential new
family members, arose once again as a derivative of the PC/Gene suite. Inspired by the
sequence analysis primer, Of URFs and ORFs (Doolittle, 1986), Bairoch began to amass
examples of short sequences, characteristic of particular binding and active sites, and

17
developed a program to scan his growing collection of sequence patterns. This part-
program, part-database chimera he named PROSITE (Bairoch, 1991). In March 1988, as part
of PC/Gene, the first release of this new resource contained 58 entries.
As with Swiss-Prot before it, PROSITE swiftly gained popularity. Its growing band of users
began not only to suggest additional patterns that could be included in the database, but
also to pressure Bairoch into giving PROSITE an independent life of its own, outside
PC/Gene. Consequently, the availability of a new public version was announced in October
1989, and formally released the following month with 202 entries (version 4.0).
Diagnostically, it was clear that sequence patterns had certain limitations. In particular,
matching a pattern is a binary match/no-match event: even the most trivial difference (a
single amino acid) results in a mis-match. As Swiss-Prot expanded and accommodated more
and more divergent members of its various superfamilies, the more evident this particular
weakness became. One solution to this problem emerged in the form of position-specific
weight matrices, or profiles. Built from comprehensive sequence alignments, profiles are
tolerant both of amino acid substitutions and of insertions/deletions; they therefore allow
the relationships between families of sequences to be modelled more realistically.
Accordingly, with the help of Phillipp Bucher, Bairoch began to augment PROSITE with
sequence profiles the first release to include them came with version 12.0, in June 1994
(Bairoch & Bucher, 1994).
Another solution, which arose (at least methodologically) independently from PROSITE,
was the development of protein family fingerprints. Fingerprints are groups of conserved
motifs, evident in multiple sequence alignments, whose unique inter-relationships provide
distinctive signatures for particular protein families and structural/functional domains.
They are diagnostically more powerful and flexible than patterns, because they can tolerate
mis-matches at the level both of individual motifs and of the fingerprint as a whole.
Fingerprints formed the basis of a database that began life as the Features Database, part of
the SERPENT information storage and analysis resource for protein sequences established at
the University of Leeds (Akrigg et al., 1992). Its first release, in October 1991, contained 29
entries: two thirds of these were linked to equivalent entries in PROSITE, which by then
held 441 family descriptions.
Although disparate in size, the Features and PROSITE databases had various aspects in
common; most notable amongst these was the principle of added-value through hand-
crafted annotation of their diagnostic signatures. In March 1991, Bairoch met Terri Attwood
for the first time at the British Crystallographic Association spring meeting in Sheffield.
Faced with the same, relentlessly time-consuming, manual-annotation burdens, they shared
their woes and discussed the wisdom of unifying the PROSITE and Features databases.
Motivated by common ideals, they later formalised their ideas in the guise of their first
European grant proposal to merge their databases into an integrated protein family
annotation resource. This was 1992; they were not successful.
In the meantime, inspired by PROSITE, a range of other signature databases began to
emerge. One of the earliest of these was Blocks, first described by Steve and Jorja Henikoff
in December 1991 (Henikoff & Henikoff, 1991). Later came ProDom (Sonnhammer &
Kahn, 1994), and later still Pfam (Sonnhammer et al., 1997). Initially linked closely to the
annotation of predicted proteins from genomic sequencing of Caenorhabditis elegans, Pfam
was to become one of the most widely used protein family databases across Europe and
the USA.


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3.8 The European Bioinformatics Institute (EBI)
Notwithstanding the proliferation of databases in the 80s, funding for their maintenance
was becoming a significant problem. By the early 90s, supporting the EMBL data library
was becoming increasingly difficult, and there was growing awareness that a more efficient
European bioinformatics infrastructure would be needed to sustain it in future. In 1992, the
EMBL concluded that the most robust solution would be to establish a new outstation,
devoted to bioinformatics. The vision of creating a European Bioinformatics Institute (EBI)
quickly took hold and, in December that year, the EMBL Governing Council published a call
for proposals to host the new facility. The deadline was extremely short (February 1993);
despite the interest of many countries, therefore, few were able to submit bids in time.
In a study by PA Consulting Group, commissioned by the ECs DGXII, a plan had been
developed for a European Nucleotide Sequence Centre (ENSC). The EMBL Council decided
to negotiate with the EC for the inclusion of the ENSC within the EBI; the EBI would provide
bioinformatics services for European scientists, be a home for the Data Library, and include
expansions in research and development necessary for long-term viability and strengthening of
neglected areas such as user support(Philipson, 1992).
In EMBLs proposal for an EBI from October 1992, worries were expressed that Europe was
lagging behind the USA: Over the last decade increments in US support for such resources have
far outstripped those in Europe, and the EBI was conceived to ensure that European research
needs are satisfied in a way which is appropriate to this global competitive context (EMBL, 1992).
The need for supportive relations between EBI and the European scientific community was
emphasised, as It would be impossible and undesirable for the EBI to be the sole bioinformatics
resource in Europe. It was noted that support should be given to major European interest
groups such as software developers, database hosts and other bioinformatics institutes; more
specifically, In recognition of the need for strong national bioinformatics activities, the EBI will
give technical and organisational support to the EMBnet Nodes, as is currently done by the EMBL
Data Library(EMBL, 1992).
Among the bidders for the EBI were Germany, Sweden and the UK. Very favourable
conditions were offered from all three. The Swedish bid for an EBI close to Uppsala
Biomedical Centre, included, for example, sufficient office space, free of rent, and high-
speed network connections. But Michael Ashburner led a more compelling UK bid. The
proposal was to host the EBI on a park, newly purchased by the Wellcome Trust, at Hinxton,
on the outskirts of Cambridge. The Trust and MRC had agreed each to fund half of the
initial capital costs of creating a complete genomics infrastructure on this site, which would
also include the newly established Sanger Centre (which, by then, had become embroiled in
the HGP) and the Human Genome Mapping Project Resource Centre (Dickson & Abbott,
1993). With its clear commitment from all levels of the UK scientific community and Government,
the UK bid won over both Uppsala and the alternative location in Heidelberg, directly
adjacent to the EMBL; it was accepted by Council in March 1993. Paulo Zanella (who had
directed the CERN Data Handling Division) was subsequently appointed as EBIs first
director (Bairoch, 2000).
The EBI became fully operational after completion of the new building in September 1995
this will no doubt have come as a great relief to the EMBL data library group, who had been
accommodated in portable cabins on the Hinxton site since the end of 1994! The new facility
had 3 broad divisions: research, industry and services, the latter being mostly devoted to
provision and maintenance of the EMBL data library and Swiss-Prot (Bairoch, 2000). The
EBIs mission was to ensure that the growing corpus of data from molecular biology and

19
genome research was placed in the public domain and was freely accessible to the entire
scientific community in order to promote scientific progress. Today, with its original 3-fold
structure still largely in place, the Institute builds, maintains and disseminates databases
and information services relevant to molecular biology, genetics, medicine and agriculture,
and undertakes leading-edge research in bioinformatics and computational biology.
Despite its pivotal role as Europes main bio-database provider, four years later, the EBI was in
financial trouble. While the Wellcome Trust and MRC had financed the initial capital costs, the
Institute relied on the EU for almost half its budget. In March 1999, however, the member
states had advised the Commission that core funding and operational costs for infrastructure
should not qualify for funding; the EBIs application for Framework funds was consequently
rejected for being out of scope. Graham Cameron, by then joint Head of the Institute with
Michael Ashburner, was quick to point out that without an immediate solution, "we will have to
abandon major projects like the DNA database, the draft human genome, the macromolecular structure
database and the microarray expression database" (Butler, 1999). The EBI was in a tricky situation,
and Britain had shot itself in the foot: it could hardly contest the Commissions ruling against
supporting the EBI because, a Commission official pointed out, it was among the countries most
against funding infrastructure directly (Butler, 1999). The situation was neatly summed up in an
editorial Nature ran at the time, If this Kafkaesque affair has any merit, it is that it has exposed the
absence of a clear mechanism for the planning and support of research infrastructure at the European
level (Nature Editorial, 1999). The cries for new mechanisms for infrastructural support, with
stable partners, stable financing and long-term political commitment, doubtless helped to sew
the seeds that in 2008 grew into the preparatory phase of ELIXIR, the European Life Science
Infrastructure for Biological Information project.
3.9 Global data overload
The late 80s and early 90s were fertile years, giving rise to a flourishing number of new
molecular structures and sequences, to new breeds of protein family signatures, and to new
databases in which to store them. Looking back at this period of fervent activity, its
incredible to reflect that two major developments had yet to take place: together, these
would not only seed an overwhelming explosion of biological data but would also spur
their global dissemination they were the advent of the Web and the arrival of high-
throughput DNA sequencing. The latter made whole-genome sequencing practically
feasible for the first time. Seizing this opportunity, there followed an unprecedented burst of
sequencing activity, yielding, in quick succession, for example, the genomes of Haemophilus
influenzae and Mycoplasma genitalium in 1995 (Fleischmann et al., 1995; Fraser et al., 1995), of
Methanococcus jannachii and Saccharomyces cerevisiae in 1996 (Bult et al., 1996; Goffeau et al.,
1996), of Caenorhabditis elegans in 1998 (C.elegans sequencing consortium, 1998), of Drosophila
melanogaster in 2000 (Adams et al., 2000) and, the ultimate prize, of Homo sapiens in 2001
(Lander et al., 2001; Venter et al., 2001; IHGSC, 2004). Hundreds of genomes have been
sequenced since this fruitful dawn.
Hand-in-hand with these activities came the development of numerous organism-specific
databases to store the emerging genomic data: for example, FlyBase (Ashburner & Drysdale,
1994), ACeDB (Eeckman & Durbin, 1995), SGD (Cherry et al., 1998), TAIR (Huala et al., 2001),
Ensembl (Hubbard et al., 2002), DictyBase (Kreppel et al., 2004) and, of course, many more. For
some, the value of this genomic gold rush was not entirely clear: with much of the amassed
data seemingly impossible to characterise, and vast amounts of it non-coding, the hoped-for


20
treasure troves were beginning to look about as inspiring as large-scale collections of
butterflies (Strasser, 2008), and perhaps suggested that molecular biology had entered a
somewhat vacuous era of high-tech stamp collecting (Hunter, 2006). Arguments like this
characterised some of the early opposition to the establishment of GenBank, and to the
substantial resistance to the Human Genome Project (HGP) a few years later (Strasser, 2008).
Perhaps inevitably, then, the HGP was an extraordinarily high-profile affair. This was partly
for the reasons outlined above, coupled with its considerable price-tag (estimated at $3 billion
from 1990-2003), but in part also because of the public-private race between Francis Collins
(who was directing the NIH National Human Genome Research Institute contributions to the
HGP) and Craig Venter (then Head of Celera Genomics) to obtain the first rough draft of
mans genetic blueprint. This intensely political drama had been preceded by a similar
struggle to be the first to sequence Drosophila, which served as a kind of warm up battle for
the human genome (Ashburner, 2006); it also had an intriguing parallel in the competition
between two public-private corporations to sequence the genome of the commercially valuable
Agrobacterium tumefaciens (Goodner et al., 2001; Wood et al., 2001; Harvey & McMeekin, 2004).
The principal tension between these public and private, and public-private hybrid, enterprises
arose not just from the race to be first to complete the sequencing: the struggle was as much
about making the results public, on the one hand, and obtaining the property rights (for
commercial exploitation, including gene patenting), on the other. Like the concerns in the early
80s surrounding NBRFs proprietary interest in protein sequences culled from the public
domain, such conflicts raised serious questions about the duty of public science to ensure that
genome sequences were made available for the public good; moreover, they challenged such
wasteful competition, resulting in the acquisition of duplicate data-sets and, usually, back-to-
back publications in high-profile journals (Harvey & McMeekin, 2004).
Another, more tangible, consequence of this intense orgy of genomic sequencing was the
generation of more data than could realistically be managed and annotated by hand and
this was just the tip of an enormous future iceberg. As illustrated in Figure 4, with each

Fig. 4. Growth of the EMBL data library (millions of entries) since its inception (red curve).
Also shown are the corresponding growth of the manually-annotated Swiss-Prot (green
line), and of structures deposited in the PDB (this line is too small to be visible!).

21
passing year from the mid 90s, there was a widening gulf both between the volume of
accumulating uncharacterised genomic sequence data and the fraction of this that it was
possible to annotate, and between the quantities of deposited biomolecular sequence and
structure data. Against this backdrop, Bairoch announced the development of a separate,
automatically generated counterpart to augment Swiss-Prot, to help disseminate the fruits of
the increasingly abundant genome projects more efficiently, without compromising the
quality of Swiss-Prot by including within it substantial quantities of uncharacterised data.
3.10 TrEMBL
By 1996, the first shock-waves from the impact of whole genome sequencing were beginning
to be felt. The aftermath was greatest for databases whose maintenance involved significant
amounts of manual annotation. Some did not recover. Swiss-Prot did survive the quake, but
to do so, new processes had to be put in place.
At the time, Swiss-Prot had the highest standard of annotation of any publicly available
protein sequence database: from the outset, one of its leading goals was to provide critical
analyses for all of its constituent sequences. To this end, each entry was accompanied by a
significant amount of annotation, derived primarily from original publications and review
articles by an expanding group of curators, with occasional input from an international
panel of experts. This high degree of meticulous manual annotation had always been the
rate-limiting step for each release of the resource; however, faced with the increased data
flow from the growing number of genome projects, this hugely labour-intensive process
simply became untenable.
To keep up, it was clear that a new approach was needed. The products of genomic
sequences had to be made available more swiftly; but how could this be achieved without
compromising the high quality of the existing Swiss-Prot data, or eroding the editorial
standards of the database in future? The answer was to prepare a computer-generated
supplement, with entries in a Swiss-Prot-like format, derived by translation of coding
sequences in the EMBL library this was TrEMBL, first released in October 1996 (Bairoch &
Apweiler, 1996). TrEMBL 1.0 contained almost 105,000 entries, not far off twice the size of
Swiss-Prot 34.0 (59,000 entries), with which it was released in parallel.
Initially, TrEMBL was an unannotated supplement to Swiss-Prot. Over the years, however,
to accelerate the process of upgrading TrEMBL entries to the Swiss-Prot standard, automatic
protocols have been established to annotate sequences with information about their
potential functions, metabolic pathways, active sites, cofactors, binding sites, domains,
subcellular location, and so on. Such information was derived from similarity and motif
searches, initially using patterns, profiles, fingerprints and so on from databases like
PROSITE, PRINTS and Pfam, and later using the amalgamated protein family resource,
InterPro. By February 2011, with many millions of entries, TrEMBL was almost 26 times
larger than Swiss-Prot, illustrating the vast disparity between manual and computer-
assisted annotation strategies.
3.11 InterPro
Rolf Apweiler was to spearhead the development of TrEMBL at the EBI in collaboration
with Bairoch at the Swiss Institute of Bioinformatics (SIB). In 1997, Michael Ashburner (then
Director of the EBI) awarded Attwood an EBI Visiting Fellowship. This entailed weekly
visits from London, and led to frequent discussions between Apweiler, Attwood and
Bairoch about sequence annotation. The feasibility of uniting PROSITE and PRINTS again


22
reared its head, but this time primarily as an instrument to help analyse and functionally
annotate the growing numbers of uncharacterised genomic sequences. Compared to the
original proposal in 1992, the case was much stronger, especially as there were now other
related databases to bring into the picture: Daniel Kahn had released ProDom in 1994, and
Richard Durbin had just announced Pfam. A new proposal was therefore submitted to the
European Commission, and the vision of an integrated protein family database was finally
funded.
In October 1999, a beta release of the unified resource was made with 2,423 entries
(representing 615 domains, 1776 families, 27 repeats and 8 sites of PTM), based on Swiss-
Prot 38.0 and TrEMBL 11.0 this was InterPro (Apweiler et al., 2001). By that time, PROSITE
and the Features Database had both undergone significant changes: PROSITE had seen 3-
fold growth to 1,370 entries (release 16.0); meanwhile, the Features Database had grown 40-
fold to 1,157 entries (release 23.1) and had been renamed PRINTS (Attwood et al., 1994).
The first release of InterPro therefore combined the contents of PROSITE 16.0 and PRINTS
23.1; it also incorporated descriptors from 241 profiles, together with 1,465 hidden Markov
models from Pfam 4.0.

Fig. 5. Stylised illustration of the relationship between the InterPro integrating hub, its
founding databases and its later additional partners, all of which contribute diagnostic
signatures and, in some cases, protein family and domain annotation. The arrows indicate
that information is shared both between satellite databases and between satellites and the
central hub. See Table 3 for further details.

23
ProDom, although part of the original consortium (see Figure 5), was not included in the
first release, initially because there was no obvious way of doing so. ProDom is built from
automatically generated sequence clusters: it isnt a true signature database, in the sense that
it doesnt exploit diagnostic discriminators; moreover, its sequence clusters need not have
precise biological correlations, so can change between database releases. Assigning stable
accession numbers to its entries was therefore impossible; this issue had to be addressed
before it could be meaningfully included in InterPro. Other factors rendered a step-wise
approach to the development of InterPro desirable. The scale of amalgamating just
PROSITE, PRINTS and Pfam was immense. Trying to sensibly merge apparently equivalent
database entries that, in fact, defined specific families, domains within those families, or
even repeats within those domains, presented enormous challenges. In the beginning,
InterPro therefore focused on amalgamating databases that offered some level of annotation,
to facilitate the integration process.
Over the years, further partners joined the InterPro consortium, as illustrated in Figure 5.
Today, with 12 primary sources, the integration challenges are legion (some of the
complexity can be understood from the list of partners, and the numbers of their signatures
that InterPro has incorporated, shown in Table 3)! With 21,185 entries in February 2011
(release 31.0), it is the most comprehensive integrated protein family database in the world
(Hunter et al., 2009).

Signature Database Version Signatures Integrated Signatures
GENE3D 3.3.0 2,386 1,377
HAMAP 021210 1,675 1,429
PANTHER 7.0 80,933 1,777
PIRSF 2.74 3,248 2,791
PRINTS 41.1 2,050 2,009
PROSITE patterns 20.66 1,308 1,292
PROSITE profiles 20.66 901 877
Pfam 24.0 11,912 11,465
ProDom 2006.1 1,894 1,008
SMART 6.1 895 882
SUPERFAMILY 1.73 1,774 1,154
TIGRFAMs 9.0 3,808 3,796
Table 3. InterPro release 31.0, February 2011.
3.12 UniProt
The year 2004 marked a turning point for the way in which protein sequence data were to be
collected and disseminated globally. The PIR-PSD, which had evolved from Dayhoffs Atlas,
had been available online since 1986; Swiss-Prot, which originally built on PIR data, also
became available in 1986; and TrEMBL had been released in 1996. The ongoing maintenance
of these disparate resources over so many years had posed major funding headaches. For
PIR, some of the difficulties were mitigated, at least in the early years, by charging for copies
of their databases and for online access to their software; later, the international
collaboration with MIPS and JIPID, supported by NSF and European grants, no doubt
helped to sustain the resource.


24
Swiss-Prot, meanwhile, had had a rocky ride and had had to be rescued from the brink of
closure, following a procedural catch-22 catastrophe: viewing Swiss-Prot as an
international resource, the Swiss government declined to provide further support unless the
database also gained a financial injection from a European Union (EU) grant; a joint
proposal with the EBI for an EU infrastructure grant, however, was declined because Swiss-
Prot was not being supported by the Swiss government! In May 1996, with only 2 months of
salary remaining for the Swiss-Prot entourage, an Internet appeal was launched announcing
the forthcoming closure, on 30 June, of Swiss-Prot and its associated databases and software
tools, owing to lack of funding. This appeal stimulated a storm of protest on the Internet, in
high-profile academic journals, and in the media. Such was the barrage that the Swiss
government stepped in, offering interim funding until the end of the year. In the
negotiations that followed, the need to create a stable vehicle for long-term funding both of
Swiss-Prot and of the Swiss EMBnet Node was discussed, and resulted in the drafting of
outline plans to establish a Swiss Institute of Bioinformatics (Bairoch, 2000).
Against this background, in 2002, with multinational funding from NIH, the NSF, the Swiss
federal government and the EU, Swiss-Prot, TrEMBL and the PIR-PSD joined forces as the
UniProt consortium. In forming the consortium, the idea was to build on the partners many
years of foundational work, by providing a stable, high-quality, unified database. This
would serve as the worlds most comprehensive protein sequence knowledgebase, replete
with accurate annotations and extensive cross-references, and accompanied by freely-
available, easy-to-use querying interfaces.
Under its hood, UniProt initially consisted of 3 separate database layers: the UniProt
Archive (UniParc), to provide a complete, non-redundant collection of all publicly available
protein sequence data; the UniProt Knowledgebase (UniProt), consisting of Swiss-Prot and
TrEMBL, to act as the central database of protein sequences, with accurate, consistent and
rich sequence and functional annotation; and the UniProt NREF databases (UniRef), to
provide non-redundant subsets of the UniProt Knowledgebase, for efficient database
searching (Apweiler et al., 2004). By 2011, UniProt also included a Metagenomic and
Environmental Sequence component, termed UniMES (The UniProt Consortium, 2011); by
this time, UniProtKB:Swiss-Prot contained 525,207 entries, accompanied by
UniProtKB:TrEMBL, with a staggering 13,499,622 entries.
3.13 The Swiss Institute of Bioinformatics (SIB)
Like the EBI, the need for which largely grew out of high-level negotiations to try to put the
EMBL data library on a more stable financial footing, the Swiss Institute of Bioinformatics
(SIB) grew out of similar high-level negotiations to establish long-term financial support for
Swiss-Prot. At the time of the Swiss-Prot funding crisis, Bairoch was aware that the Swiss
scientific authorities had been emphasising the need to establish centres of excellence in
economically important, interdisciplinary areas that would be crucial for tomorrows
society. Seizing upon this, together with Ron Appel, Philipp Bucher, Victor Jongeneel and
Manuel Peitsch, he submitted a proposal to create a Swiss bioinformatics institute, whose
goals were to:
promote the development of bioinformatics software tools and databases;
sustain high-quality bioinformatics research;
collaborate with academic partners to provide a curriculum to train research scientists
in the field of bioinformatics; and
offer services to its user community through the Swiss Node of EMBnet.

25
After a lengthy period of consultation, the SIB was finally created as a non-profit foundation
in March 1998, with Victor Jongeneel as the first director. The founders then went on to win
funds for some of the SIBs activities from the Swiss Federal government: by law, only 50%
of the Institutes work could be funded in this way the rest had to come from other
sources, preferably by commercial exploitation of its research.
Partly in response to this stipulation, but partly also because it had become clear that Swiss-
Prot could not be reliably sustained solely with public funding, the decision was made to
ask commercial users of the database to pay a licence fee. Various models for achieving this
were tested; in the end, in 1997, Bairoch, Appel and Denis Hochstrasser decided that the best
way forward was to set up a new company this was Geneva Bioinformatics SA (GeneBio).
Up to three quarters of the revenues now generated by GeneBio from sales of annual
database and software licences are returned to SIB, thereby helping to bolster the work of
the Swiss-Prot groups (Bairoch, 2000).
Today, the SIB leads and coordinates the field of bioinformatics in Switzerland: its vision, to
help shape the future of the life sciences through excellence in bioinformatics services,
research and education; its mission, to provide world-class core bioinformatics resources to
both national and international research communities in fields spanning genomics,
proteomics and systems biology. Many of its core activities, including maintenance of
databases such as UniProt and InterPro, are carried out in close collaboration with the EBI.
3.14 The European Nucleotide Archive (ENA)
Meanwhile, with the advent of large-scale sequencing projects and the dawn of Next
Generation Sequencing (NGS) technologies, a mounting tsunami of nucleotide sequence
data was growing force across the globe; a number of important developments were to take
place in its wake. By 2003, it was clear that there was a need to provide access not only to the
most recent versions of sequences, but also to their historical artifacts following the rush to
patent genetic information, issues of priority became increasingly important, and it was vital
to be able to see sequence entries exactly as they appeared in the past. Accordingly, the EBI
established a Sequence Version Archive (Leinonen et al., 2003), to store both current and
earlier versions of entries in the EMBL data library (which, by then, had been dubbed
EMBL-Bank).
By September 2004, EMBL-Bank had grown prodigiously, with more than 42 million entries
(Kanz et al., 2005) and, by 2007, was accompanied by the Ensembl Trace Archive (ETA) the
ETA was set up to provide a permanent archive for single-pass DNA sequencing reads
(from whole-genome shotgun, EST and other large-scale sequencing projects) and associated
traces and quality values. Together, EMBL-Bank and the ETA became known as ENA, the
European Nucleotide Archive, Europes primary nucleotide-sequence repository (Cochrane
et al., 2008). Throughout 2007, ENA continued to grow in terms both of its volume and of the
nature of data it contained such that, by October of that year, it included more than 1.7
billion records (comprising ~1.7 trillion (1.7x10
12
) base pairs of sequence) (Cochrane et al.,
2008). By 2010, ENA had embraced a third component the Sequence Read Archive (SRA)
and now contained ~500 billion raw and assembled sequences, comprising 50x10
12
base
pairs; this is a phenomenal growth in just 3 years! During this period, NGS reads held in the
SRA had become the largest and fastest growing source of new data, and accounted for
~95% of all base pairs made available by ENA (Leinonen et al., 2011). Contributing to this


26
mass of data were the completed genomes of more than 1,400 cellular organisms, and 3,000
viruses and phages.
But such enormous progress comes at a cost, challenging current IT infrastructures to the
limit. Some of the oldest data in ENA date back to the early 80s, with the inception of the
EMBL data library. As an aside, it is somewhat ironic that, even in those days, there were
distribution headaches. Bairoch, for example, relates how difficult it was to transfer
version 2 of the EMBL data library from computer tape to a mainframe computer and
thence to his microcomputer, because the mainframe had no communication protocol to
talk to a microcomputer he therefore had to spend the night transferring the data, screen
by screen, using a 300 baud acoustic modem (Bairoch, 2000). To put this in perspective,
this version of EMBL-Bank contained 811 nucleotide sequences (with more than 1 million
base pairs) this is about the same amount of data that currently enters ENA every 2
seconds.
Today, ENA holds more than 20 terabases of nucleotide sequence data, which, combined
with its annotation information, and so on, occupies more than 230 terabytes of disk
space. The infrastructure required to store, maintain and service such a vast archive, and
the cost of doing so, is beyond anything that either the originators of the first databases,
or the developers of the new sequencing technologies could have conceived. Interestingly,
in February 2011, the NCBI announced that it would be discontinuing its Sequence Read
and Trace Archives for high-throughput sequence data, owing to budget constraints. The
closure of the databases is to be phased, and completed within 12 months. The NCBI is
still committed to supporting and developing information resources for biological data
derived from NGS technologies (genotypes, variations, assemblies, gene expression data,
and so on), but will need to find new funding strategies for access to and storage of the
existing data.
3.15 ELIXIR
The opportunities NGS technologies present for advancing life science research (especially
in areas such as healthcare, food security, energy diversification and environmental
protection) are incredibly exciting; but these opportunities will be lost if they are not
underpinned by a robust, effective and sustainable information infrastructure. The best
estimates today suggest that, by 2020, NGS technologies will be producing data at up to a
million times the current rate. Development of an appropriate infrastructure to manage the
data deluge is therefore paramount.
The ELIXIR project is the realisation of this urgent need. Recognising that the task is of such
magnitude that it cannot be tackled by a single organisation, it is a call to arms for
international cooperation in building a pan-European infrastructure to help extract the
maximum value from the investments that have already been made, and from those that
will be made in future, in this area. The plan is for the ELIXIR infrastructure to be
distributed across a variety of Nodes hosted by centres of excellence across Europe, and for
each of these to be connected to the EBI central Hub. It is expected that some of the Nodes
will act as national coordination centres to expedite interactions both with the Hub and with
local funders; Nodes that perform similar functions will be expected to collaborate to form
ELIXIR service networks, providing data or compute resources, or training, according to
their speciality, as depicted in Figure 6.

27

Fig. 6. Proposed topology of the ELIXIR Hub and Nodes. In an arrangement reminiscent of
EMBnet 23 years before it, some of the Nodes are expected to serve as national
bioinformatics centres; others, with similar functions, will collaborate as service networks,
for example to provide data or compute resources, or training.
Initially, the numbers of Nodes is expected to be small, growing to ~20 during the first 5
years of the initiative (during the preparatory phase, more than 50 institutions submitted
expressions of interest in becoming ELIXIR Nodes), at a cost of several hundred million
euro. To garner support for the business case, governments of the European Member States
have been invited to sign a non-binding Memorandum of Understanding (MoU) in order to
initiate negotiations to construct ELIXIR; the MoU will become effective once 5 countries
and the EMBL have signed. Europes databases (estimated to number around 500),
especially those hosted by the EBI, will become the foundation of the new ELIXIR
infrastructure as part of its mission, to construct and operate a sustainable infrastructure for
biological information in Europe to support life science research and its translation to medicine and
the environment, the bio-industries and society (Thornton, 2011).
4. The development and spread of tools to keep pace with the new
technologies
With the sequencing of biopolymers and subsequent organisation of the growing mass of
biosequences in databases, visual comparison techniques became tedious, not least because


28
the determination of the significance of a given result usually is left to intuitive rationalization
(Needleman & Wunsch 1971). To reduce reliance on manual (often subjective) interpretation
and put sequence analysis on a more systematic footing, algorithms to analyse and compare
sequences began to emerge. As early as 1966, Fitch proposed computational analysis to
study evolutionary homology, using mutation values to indicate how many nucleotides in
the genomic code must change in order to introduce change (mutation) at the amino acid
level. In 1970, Needleman and Wunsch described the first algorithm to quantify the
similarity between two protein sequences (so-called global alignment) today, this
algorithm is still used to identify similarities between two sequences and infer likely
ancestry. Years later, Smith and Waterman (1981) presented an algorithm to find local
similarities: to find a pair of segments, one from each of two long sequences, such that there is no
other pair of segments with greater similarity. In time, more efficient methods were required to
compare newly sequenced proteins against the rapidly expanding databases. FASTP was
the first fast algorithm (Lipman & Pearson 1985).
Search algorithms like this afforded many of the earliest and most exciting discoveries
attributable to bioinformatics. For example, one of the first observations that gave a clue to
the molecular mechanism of neoplastic transformation was provided by the finding of a
near identity in amino acid sequence between the platelet-derived growth factor (PDGF) B-
chain and a region in the transforming protein, p28sis, of simian sarcoma virus (SSV), an
agent that causes sarcomas and gliomas in experimental animals (Waterfield et al., 1983).
This finding arose from computer searches using the Wilbur and Lipman algorithm on the,
at the time (1983) available, NEWAT protein database created by Doolittle et al. This first
success story, where simple sequence comparison led to the completely new concept of
gene-oncogene, showed the medical community the enormous potential of computer
techniques for sequence comparison and analysis.
In a similar way, DNA sequencing having been revolutionised by Sanger and by subsequent
improvements of his technique, and having given rise to the growing number of nucleotide
sequences being collected in data repositories like the EMBL data library and GenBank, so
too algorithms to search these databases became a necessity. FASTA was a more sensitive
modification of FASTP, and had the advantage of being able to search nucleotide sequence
databases with either a nucleic acid or protein sequence by translating the DNA database
during the search (Pearson & Lipman 1988). Later, somewhat overshadowing these
developments, came the Basic Local Alignment Search Tool, BLAST (Altschul et al., 1990);
this offered an extended tool-set to apply any kind of sequence database search, and is still
the most widely used tool in bioinformatics. The success of BLAST spawned a number of
more specialised sequence search methods, such as PSI-BLAST, PHI-BLAST, BLAT, and so
on, and is itself still in continuous development (Camacho et al., 2009).
Aside from these very popular database search tools, many other sequence, annotation and
expression analysis tools were developed for a broad range of applications: e.g., for pattern
recognition, for protein and RNA secondary structure prediction, for microarray data
analysis, for proteome and genome annotation, and so on. In the early 90s, building on the
existing University of Wisconsin Genetics Computer Group (UWGCG, or simply GCG)
package, several such algorithms were collected at the EMBL and packaged as GCGEMBL
Utilities, later known as Extended GCG. However, GCG was then commercialised and its
distribution policy changed. Reacting against the new policies, in 1998 several software
developers founded EMBOSS, the European Molecular Biology Open Software Suite. Their

29
aim was to develop new sequence analysis tools, by replacing popular but obsolete EGCG
applications, and integrating with SRS, ACEDB, and a range of other publicly available
software interfaces and tools. The idea was to encourage other developers to use the
EMBOSS software libraries, and especially to harness the expertise and potential additional
manpower at EMBnet Nodes (e.g., in Germany, Italy, France, The Netherlands, Austria,
Russia, Switzerland, Israel, Spain, Norway, and so on). Target users of the resource included
those at the Sanger Centre, those served by EMBnet, and those in academic and
pharmaceutical settings. Funded by the Wellcome Trust for 3 years, the project was a
collaborative effort of the Sanger Centre, EMBnet UK (SEQNET), the EBI and CNRS
Montpellier.
With the pivotal support of EMBnet, EMBOSS quickly became a comprehensive
bioinformatics resource (Rice et al., 2000). There are now several incarnations of the suite
with different GUIs, including the EMBOSS teams Java-based interface, jEMBOSS; the
Belgian and Argentinian EMBnet Nodes wEMBOSS; and the EMBOSS GUI from the
National Research Council of Canada. Today, EMBOSS is still being developed, adopting
new specific file formats and algorithms in order to embrace the world of NGS data
analysis.
Another important development driven by the EMBL was the Sequence Retrieval System
(SRS), an information indexing system applied to flat-file databases, such as the EMBL data
library, Swiss-Prot and PROSITE (Etzold and Argos, 1993). SRS became the most widely
used data-retrieval system for flat-file systems, with an extended GUI to extract not only
sequences but all related information, via an exhaustive sequence query and export system
(Zdobnov et al., 2002).
Europe-wide, there are vast numbers of other specialised biological data-analysis, data-
visualisation and data-retrieval tools available: many of these are provided by the EBI;
others by the SIBs ExPASy Proteomics Server; some are offered via the National and
Specialist Nodes of EMBnet; others are available as Web services collected in the
BioCatalogue (Bhagat et al., 2010). The BioCatalogue evolved from the EMBRACE registry
(Pettifer et al., 2010), one of the end products of the EMBRACE project (European Model for
Bioinformatics Research and Community Education) this was a 5-year FP7 Network of
Excellence, whose main goal was to orchestrate highly integrated access to a broad range of
bio-molecular data and software packages. Achieving this required standardised access to
tools and databases; to this end, the decision was to use Web services. In consequence, many
of the project partners adapted their tools and database-access protocols, and logged their
Web services in a common registry. At the end of EMBRACE, in 2010, the registry was
handed over to the BioCatalogue, which is now being maintained in collaboration with
myExperiment, myGrid, seekda and BioMoby, and hosts 2,053 services from 147 service
providers and 505 members.
5. The central place of bioinformatics in modern biology
Clearly, we have travelled a very long way since Jensen and Evans positioned a single
amino acid (a terminal phenylalanine) in insulin (Jensen & Evans, 1935; Sanger, 1945;
Sanger, 1988) and Sanger elucidated its complete sequence, the first of any protein (recall
Table 2). In a story spanning something like 70 years, bioinformatics has given us the first
complete catalogues of DNA and protein sequences, including the genomes and proteomes


30
of organisms across the entire Tree of Life; it has furnished the requisite software to help
analyse biological data on an unprecedented scale; it has hence yielded the possibilities to
understand more about evolutionary processes in general, our place in the Tree of Life in
particular, and ultimately, a great deal more about health, disease and disease processes.
Figure 7 offers a summary of some of the most important landmarks that have charted the
development of bioinformatics in Europe and helped to place it at the heart of 21
st
century
biology.

Fig. 7. Historical milestones that have placed bioinformatics at the heart of 21
st
century
biology, from the determination of the first amino acid sequence, to the development of an
archive of 500 billion nucleotide sequences. Some major milestones are denoted in black; key
computing innovations are indicated in purple; example databases are indicated in blue;
organisations and institutions in green; numbers of sequences in red, the growing mass of
which is highlighted both in the red curve and the background gradient the impact of
genomic sequencing in the mid 90s is clear.
6. Conclusion European bioinformatics goes global
The history of bioinformatics has clearly been a convoluted interplay between events in
Europe, the USA, Japan and across the globe. Here, we have attempted to recount the story
primarily from a European perspective as it unfolded largely from the point of view of
sequence data: in terms of the technological innovations that spawned their extraordinary

31
growth and dissemination, of the databases that grew up to manage and analyse them, and
of the institutions and infrastructural initiatives that arose to try to give those databases
some measure of financial stability. In so doing, we accept that weve only scratched the
surface, and we regret any shortcomings that may have arisen from the necessary omission
of so many of the other important details and perspectives.
Clearly, the evolution and impact of bioinformatics reaches far beyond Europe, and there
are now many organisations world-wide with missions to bring life science data to their
local communities, to make freely available easy-to-use software tools with which to analyse
the data, and to provide training, both to users of bioinformatics databases and software,
and to new generations of bioinformatics trainers (Schneider et al., 2010). In this context,
EMBnet, for example, which began life as the European Molecular Biology Network, is now
a global bioinformatics network, maintaining fruitful cooperations with the Iberoamerican
(SoIBio) and Asia Pacific (APBioNet) bioinformatics networks, as well as with the USA-
based International Society for Computational Biology (ISCB); it has also established close
ties with the African Society for Bioinformatics and Computational Biology (ASBCB), and
synergies with other relevant groups in northern Africa are now developing. Interestingly,
33 years ago, Joshua Lederberg observed that, the claim of science to universal validity is
supportable only by virtue of a strenuous commitment to global communication (Lederberg, 1978).
Today, this is a commitment that EMBnet vigorously pursues; in a similar spirit, we can be
quite sure that the contribution of Europe to the future evolution of bioinformatics will
continue in a global arena.
7. Acknowledgement
We would like to thank Vicky Schneider for providing the inspiration (and the title) for this
chapter.
8. References
Adams, M.J.; Blundell, T.L., Dodson, E.J., Dodson, G.G., Vijayan, M., Baker, E.N., Harding,
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Part 2
Data Integration

2
Data Integration in Bioinformatics:
Current Efforts and Challenges
Zhang Zhang
1
, Vladimir B. Bajic
1
, Jun Yu
2
,
Kei-Hoi Cheung
3,4,5,6
and Jeffrey P. Townsend
6,7

1
Computational Bioscience Research Center (CBRC),
King Abdullah University of Science and Technology (KAUST), Thuwal
2
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics,
Chinese Academy of Sciences, Beijing
3
Center for Medical Informatics,
4
Department of Computer Science,

5
Department of Genetics,
6
Program in Computational Biology and Bioinformatics,
7
Department of Ecology and Evolutionary Biology, Yale University,
New Haven, Connecticut
1
Kingdom of Saudi Arabia
2
China
3,4,5,6,7
United States of America
1. Introduction
With the rapid advancements in next-generation sequencing (NGS) technologies and the
consequently fast-growing volume of biological data, a diversity of data sources (databases
and web servers) have been created to facilitate data management, accessibility, and
analysis. A prerequisite of bioinformatics research has been the ability to find, maneuver
and access data deposited in various data sources. For a given bioinformatic task,
researchers often need to be skillful in interrogating these data sources, and in the use of
extracted information for further data analysis/information search. For example, one must
obtain data from one data source, reformat the data and submit to another data source for
analysis, parse the analyzed result, and then combine the result with data obtained from the
third data source, etc. Undisputedly, data integration becomes tedious and time-consuming,
especially regarding the import and export of enormous files of modern NGS and other
data. Thus, integration of data from distributed, heterogeneous and voluminous data
sources turns out to be a significant obstacle to fully exploit the wealth of big biological data
(Davidson, et al., 1995; Stein, 2002). The importance of the integration component of research
stemming from studies based on high-throughput technologies (such as NGS), is twofold:
(1) due to the great level of automation of the actual experimental procedures, the effort of
obtaining the experimental data takes only about 20% or less of the overall research effort in
an NGS project; approximately four fifths of the effort goes to the integration and analysis of
a collection of the experimental data (Mardis, 2010); (2) the answers to the most important,
complex biological questions today are rarely provided directly through the experimental


42
results; to bring potential answers to the surface, downstream bioinformatics analysis often
involves the integration of diverse data from multiple data sources.
The objective of data integration in bioinformatics is to establish automated and efficient ways
to integrate large, heterogeneous biological datasets from multiple sources. However, this
objective is challenged by data sources that are geographically distributed and heterogeneous
in terms of their functions, structures, data access methods and dissemination formats.
According to the 2010 update on the Bioinformatics Links Directory (Brazas, et al., 2010), there
are almost 1500 unique publicly-available data sources. Based on their functions, data sources
can be classified into diverse categories: (1) sequence databases, e.g., GenBank (Benson, et al.,
2006), RefSeq (Pruitt, et al., 2009), CMR (Comprehensive Microbial Resource) (Davidsen, et al.,
2010); (2) functional genomics databases, e.g., ArrayExpress (Parkinson, et al., 2011), FFGED
(Filamentous Fungal Gene Expression Database) (Zhang and Townsend, 2010), GEO (Gene
Expression Omnibus) (Barrett, et al., 2011); (3) protein-protein interaction databases, e.g., BIND
(Biomolecular Interaction Network Database) (Bader, et al., 2003), DIP (Database of Interacting
Proteins) (Salwinski, et al., 2004), IntAct (Aranda, et al., 2010), MINT (Molecular Interactions
Database) (Ceol, et al., 2010); (4) pathway databases, e.g., KEGG (Kyoto Encyclopedia of Genes
and Genomes) (Kanehisa, et al., 2010); (5) structure databases, e.g., CATH (Greene, et al., 2007),
PDB (Protein Data Bank) (Rose, et al., 2011); (6) annotation databases, e.g., GO (Gene
Ontology) (Ashburner, et al., 2000), NCBI Taxonomy (Sayers, et al., 2011). Moreover, data
sources differ in data accessibility and dissemination. That is, different levels of provision are
made by the data source managers for human-reading, computer-reading, or both. Certainly,
data sources can also be classified by species of interest, such as, filamentous fungi (Zhang and
Townsend, 2010), fly (Gilbert, 2007), mouse (Blake, et al., 2011), and yeast (Engel, et al., 2010).
Despite the challenges, the promise of data integration is high: heterogeneous data sources
provide biological data encompassing a wide range of research fields. Therefore, data
integration has the potential to facilitate a better and more comprehensive scope of inference
for biological studies. Although efforts have been devoted to biological data integration over
the past two decades, it remains challenging and laborious. Here we review current efforts
and illustrate several approaches used for data integration. With a specific consideration of
the exponentially-growing NGS data, we also describe challenges in this context and discuss
potential trends.
2. Current efforts of data integration in bioinformatics
Several major approaches have been proposed for data integration, which can be roughly
classified into five groups (Goble and Stevens, 2008; Zhang, et al., 2009): data warehousing,
federated databasing, service-oriented integration, semantic integration and wiki-based
integration. Across all of these groups, to a significant extent, an increasingly important
component of data integration is the community effort in developing a variety of biomedical
ontologies (see Section 3.2), to deal in a more specific manner with the technicality and
globality of descriptors and identifiers of information that has to be shared and integrated
across various resources (Antezana, et al., 2009; Maojo, et al., 2011; Rubin, et al., 2008).
2.1 Data warehousing
The data warehouse approach offers a one-stop shop solution to ease access and
management of a large variety of biological data from different data sources. Data
warehouses focus on data translation, fetching all accessible data from many disparate data

Data Integration in Bioinformatics: Current Efforts and Challenges

43
sources, transforming the data and importing it into the data warehouse. Representative
examples of data warehousing include:
Atlas (Shah, et al., 2005) is a biological data warehouse that locally stores and integrates
biological sequences, molecular interactions, homology information, functional
annotations of genes, and biological ontologies. It includes data from BIND, DIP, Entrez
Gene (Maglott, et al., 2011), GO, GenBank, HomoloGene, HPRD (Human Protein
Reference Database) (Keshava Prasad, et al., 2009), IntAct, LocusLink (Pruitt and
Maglott, 2001), MINT, RefSeq, OMIM (Online Mendelian Inheritance in Man)
(Amberger, et al., 2009), Taxonomy, and UniProt (The UniProt Consortium, 2011).
BioWarehouse (Lee, et al., 2006) is an open source toolkit for constructing data
warehouses. It incorporates data from BioCyc (Karp, et al., 2005), CMR, ENZYME
(Bairoch, 2000), GenBank, GO, KEGG, Taxonomy, and UniProt and integrates its
component databases into a common representational framework within a single
database management system.
BIOZON (Birkland and Yona, 2006) is a unified biological resource on DNA sequences,
proteins, complexes and cellular pathways. It relies on an extensive database schema
that integrates information at the macro-molecular level as well as at the cellular level
from a variety of data sources, including BIND, DIP, Genbank, InterPro (Hunter, et al.,
2009), KEGG, PDB, RefSeq, Swiss-Prot (Bairoch, et al., 2004), UniGene (Sayers, et al.,
2011), and UniProt.
COLUMBA (Trissl, et al., 2005) is an integrated database of information on proteins,
structures and annotations. It integrates twelve different databases, including CATH,
ENZYME, GO, KEGG, PDB, SCOP (Andreeva, et al., 2008), and Swiss-Prot.
VINEdb (Hariharaputran, et al., 2007) is a data warehouse for integration and
interactive exploration of life science data. It manages diverse data from GO, IntAct,
KEGG, OMIM, and UniProt and emphasizes the visualization of the integrated data in a
comprehensible manner.
The data warehouse approach has several advantages. (1) The user does not need to access
many web sites for multiple data sources. Data warehouses provide one single access point
to conveniently manipulate a large variety of data. (2) All queries requested by users are
executed within the data warehouse (rather than on distributed data sources) and therefore,
data warehousing eliminates network bottlenecks and obtains high performance with fast
response. (3) Due to data storage at a single managed point, data warehousing obtains
benefits in data control, yielding easy customization to meet users needs.
Despite its advantages, the data warehouse approach has a major problem; it requires
continuous and often human-guided updates to keep the data comprehensive of the
evolution of data sources, resulting in high costs for maintenance. In general, there are two
kinds of changes. (1) Changes in data volume or revisions of data. Whenever extant data is
revised or the volume of data in any data source is changed, the data warehouse must
monitor for such remote changes and update the warehouse to store the new data. (2)
Changes in data structure, including adding new data types and tables, changing database
tables and their relationships, and changing output formats. Many biological data sources
change their data structures roughly twice a year (Stein, 2003). Whenever the data sources
change their data structures, consequent data translation into the data warehouse must be
updated in response. Usually, modification of data translation is labor-intensive and
expensive.


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2.2 Federated databasing
Unlike data warehousing (with its focus on data translation), federated databasing focuses
on query translation. The federated databasing approach executes all queries on the
distributed sources by translating a query against the federated database into a query
against many data sources. The federated database fetches the data from disparate data
sources and then displays the fetched data for its user base. Representative examples for
federated databasing include:
BioMart (Haider, et al., 2009) is a query-oriented data integration system developed
jointly by the Ontario Institute for Cancer Research (OICR) and the European
Bioinformatics Institute (EBI). It provides a user-friendly and unified way to retrieve
data from one or multiple data sources located at diverse geographical locations,
including Ensembl (Flicek, et al., 2011), HGNC, Uniprot, Reactome (Croft, et al., 2011),
Wormbase, and PRIDE (Jones, et al., 2008).
DiscoveryLink (Haas, et al., 2001) developed by IBM is a system for integrated access to
life sciences data from heterogeneous data sources, including GenBank, MedLine and
Swiss-Prot. It features query optimization and cross-source queries that access relational
databases and retrieve the data from diverse data sources.
K2/Kleisli (Chung and Wong, 1999; Davidson, et al., 2001) is a federated database
system, integrating data from EcoCyc (Keseler, et al., 2011), GenBank, GSDB (Harger, et
al., 1998), dbEST (Boguski, et al., 1993), GDB (Letovsky, et al., 1998), KEGG and SRS-
indexed databases. Kleisli uses a high-level query language called Collection
Programming Language (CPL) as its query language, which was developed specifically
for parsing, optimizing and executing queries. K2 is the newer version of Kleisli and
replaces CPL by a powerful and easy-to-use SQL-like query language, Object Query
Language (OQL).
MRS (Hekkelman and Vriend, 2005) allows for very rapid queries in a large number of
flat-file data banks, including EMBL, UniProt, OMIM, dbEST, PDB, KEGG. It combines
a fast and reliable backend with a very user-friendly implementation of all the
commonly used information retrieval facilities.
QIS (Query Integrator System) is based on a set of distributed network-based servers,
data source servers, integration servers, and ontology servers and relies on a
combination of SQL-like syntax and XML (eXtensible Markup Language; a widely used
standard for data description and exchange), to formulate a query (Marenco, et al.,
2004). It stores diverse queries for data integration from continuously changing
heterogeneous data sources in the biosciences, including CellPropDB (Crasto and
Shepherd, 2007), Brain Architecture Management System (Bota and Swanson, 2010),
Yale Microarray Database (Cheung, et al., 2002), a local Gene Annotation Database and
GO.
SRS (Sequence Retrieval System) is an index-based integration system and combines
some features of data warehousing and federated databasing (Zdobnov, et al., 2002).
SRS uses a keyword-based indexing language ICARUS to describe each integrated data
source and locally creates a full-text index over all data sources. Meanwhile, it allows a
single query to execute on multiple data sources based on local indexed entries. SRS
contains a number of biological databases (see details in http://srs.ebi.ac.uk/
srsbin/cgi-bin/wgetz?-page+databanks+-noSession).
TAMBIS (Transparent Access to Multiple Bioinformatics Information Sources) is an
integration application to perform bioinformatics tasks over multiple data sources by


45
using an ontology of biological concepts (Stevens, et al., 2000). The prototype version of
TAMBIS contains five data sources, viz., BLAST, CATH, ENZYME, PROSITE (Sigrist, et
al., 2010) , and Swiss-Prot.
Queries in federated databases are executed within remote data sources and results
displayed in federated databases are extracted remotely from the data sources. Due to this
capability, federated databasing has two major advantages. (1) Federated databases can be
regarded as an on-demand approach to provide immediate access to up-to-date data
deposited in multiple data sources. (2) Compared with data warehousing, federated
databasing does not replicate data in data sources; therefore, it presents relatively
inexpensive costs for storage and curation. However, federated databasing still has to
update its query translation to keep pace with data access methods at diverse remote data
sources. In addition, since data is retrieved from remote data sources, federated databasing
depends heavily on network connectivity and query complexity, which may lead to low
efficiency and speed in data retrieval.
2.3 Service-oriented integration
Data warehousing and federated databasing both focus on centralizing data access, through
data translation and query translation, respectively. They confront some similar problems
stemming from data storage and curation, frequent updates, and high costs for data
exchange and/or maintenance. In part to evade these issues, a decentralized approach has
also been advanced, in which individual data sources agree to open their data via Web
Services (WS). WS are designed for communication between computers over the Web and
described by the Web Services Description Language (WSDL). There are several different
protocols for WS, e.g., SOAP (Simple Object Access Protocol; a protocol for exchanging
XML-based messages over computer networks), REST (REpresentational State Transfer; a
simple protocol implemented using HTTP methods). WS support computer-to-computer
interaction through Web Application Programming Interface (Web API) (Shi, 2007) and can
perform a database query or computation. In the context of data integration, data can be
programmatically accessed via WS and data sources serve as service providers. Therefore,
this approach can be seen as a service-oriented approach. The service-oriented approach
enables data integration from multiple heterogeneous data sources through computer
interoperability. Several representative examples for service-oriented integration include:
BioMOBY (Kawas, et al., 2006; Wilkinson and Links, 2002; Wilkinson, et al., 2008) is an
open source ontology-based integration system for accessing distributed and
heterogeneous data sources via WS. It implements a WS registry and uses standard
ontology terms to annotate WS. BioMOBY adopts SOAP for data exchange and allows
interoperability among different data sources to achieve automated data integration
and sharing (Neerincx and Leunissen, 2005).
DAS (Distributed Annotation System) is a client-server system to provide access to
complete distributed genome annotations using SOAP-based WS (Dowell, et al., 2001;
Katayama, et al., 2010; Olason, 2005). It allows a single machine to collect all annotations
from multiple distributed data sources and display them to the user in a single view.
DAS is widely used in the genome annotation community
(http://en.wikipedia.org/wiki/Distributed_Annotation_System) and adopted by
several systems, including Ensembl, WormBase, and the Berkeley Drosophila Genome
Project (Jenkinson, et al., 2008; Messina and Sonnhammer, 2009; Olason, 2005).


46
Taverna (Oinn, et al., 2004), a part of MyGrid (Stevens, et al., 2003), is a graphical
workflow workbench application, aiming to integrate the growing number of molecular
biology tools and databases (Hull, et al., 2006). Workflows in Taverna, written by a
custom XML-based language called Simple Conceptual Unified Flow Language
(SCUFL), can automatically record all data involved, provenance metadata, and results,
facilitating complex data processing in a dynamic distributed environment.
The service-oriented approach features data integration through computer-to-computer
communication via Web API and up-to-date data retrieval from diverse data sources. Thus,
it befits well with the dynamic nature of bioinformatics. However, it remains challenging,
primarily because its success in heterogeneous data integration requires that many data
sources should become service providers by opening their data via WS and by
standardizing data identities and nomenclature to ease data exchange and analysis. In
addition, a unified WS registry is also necessitated, not only to establish standards for WS
registration, but also to formulate standards for service-oriented workflows or pipelines
(Zhang, et al., 2009).
2.4 Semantic integration
Most web pages in biological data sources are designed for human reading (e.g., HTML).
The Semantic Web (Dibernardo, et al., 2008; Good and Wilkinson, 2006; Hendler, 2003; Lord,
et al., 2004) aims to describe data in a way that computers can understand and to build an
interconnected network that computers can easily and unambiguously process. According
to the statement of definition from the World Wide Web Consortium (W3C), the purpose of
the Semantic Web is to create a universal medium for the exchange of data using several
standards, including Resource Description Framework (RDF; http://www.w3.org/RDF),
RDF schema (RDFSRDF Vocabulary Description Language; http://www.w3.org/TR/rdf-
schema), Web Ontology Language (OWL; http://www.w3.org/owl), and standard Web
query language SPARQL (http://www.w3.org/TR/rdf-sparql-query) for RDF. RDF
provides standard formats (e.g, XML format) for data interchange and describes data as a
simple statement, containing a set of triples: a subject, a predicate and an object. Any two
statements can be linked by an identical subject or object. OWL builds on RDF and Uniform
Resource Identifier (URI) and describes data structure and meaning based on ontology,
which enables automated data reasoning and inferences by computers. The Semantic Web
provides an machine-readable way for data representation and interoperability (Antezana,
et al., 2009). Several studies have applied the Semantic Web technologies in data integration
and representative examples of semantic integration are described below.
Bio2RDF (Belleau, et al., 2008) is a mashup system that creates an integrated space of
RDF documents linked together with normalized URIs. Bio2RDF applies the Semantic
Web technologies to multiple data sources, such as Entrez Gene, HGNC, KEGG, MGI,
OMIM PDB, PubMed and UniProt, and converts data into RDF format based on
RDFizer (a set of tools for converting various data formats into RDF;
http://simile.mit.edu/wiki/RDFizers), Sesame (an open source framework for storage,
inference and querying of RDF data; http://www.openrdf.org) and OWL ontology. In
Bio2RDF, each RDF document is expressed as a URI. When a query is requested to
Bio2RDF for a given URI, for example, http://bio2rdf.org/go:0004396, the URI
identifies RDF triples containing the GO term of Hexokinase (GO:0004396). Bio2RDF
supports query via SPARQL.


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HCLS (The Health Care and Life Sciences Interest Group;
http://www.w3.org/2001/sw/hcls/), established by W3C, aims to explore the
potential benefits of the Semantic Web in the health care and life sciences domains
(Cheung, et al., 2008) and advocates the application of the Semantic Web for advancing
translational research (Ruttenberg, et al., 2007). The HCLS Knowledge Base (HCLS-KB;
http://www.w3.org/TR/hcls-kb) is a Semantic Web system that imports data from
many data sources in multiple domains of life sciences, including not only general
sources, e.g., Entrez Gene, GO, HomoloGene, but also domain-specific sources, e.g.,
Allen Brain Atlas (an interactive, genome-wide image database of gene expression in
the mouse brain; http://www.brain-map.org) (Lein, et al., 2007), SenseLab (a collection
of neuroscience data; http://neuroweb.med.yale.edu/senselab) (Crasto, et al., 2007)
and SWAN (Semantic Web Applications in Neuromedicine; aiming to organize and
annotate scientific knowledge about Alzheimer disease and other neurodegenerative
disorders) (Ciccarese, et al., 2008; Clark and Kinoshita, 2007; Kinoshita and Clark, 2007).
YeastHub (Cheung, et al., 2005) is an integrated database in RDF format for the yeast
community. It creates a RDF repository for RDF storage and provides a utility to
convert tabular format into RDF format. YeastHub integrates different types of yeast
data provided by different data sources (SGD, YGDP, MIPS, BIND, GO and TRIPLES)
and supports RDF-based queries to retrieve and query the data.
Application of the Semantic Web technologies to biological data integration is a significant
advancement for bioinformatics, enabling automated data processing and reasoning. The
semantic integration uses ontologies for data description and thus represents ontology-
based integration (Noy, 2004). However, the Semantic Web continues to evolve and its
application in biological data integration has several limitations. The semantic integration
locally stores a large collection of RDF documents, by copying data from multiple data
sources and converting data into RDF format. From this view, the semantic integration can
be regarded as a special data warehouse with data in RDF format. As a consequence, it
inherits the pros and cons of data warehousing and is vulnerable to updates in data sources.
To keep the RDF documents up-to-date, it requires tedious and periodical data retrieval and
RDF conversion. In addition, once any data source changes data structure, the RDF
conversion scripts must be updated consequently.
Currently, there is an ongoing project, the World Wide Web Consortium's SWEO (Semantic
Web Education and Outreach) Linking Open Data Project (Bizer, 2009; Zhao, et al., 2009)
that uses the Semantic Web technologies to connect related distributed data across the Web.
Technically, linked data rely on RDF to create typed links between data from different data
sources. Linked data is machine-readable, explicitly defined, and inter-linked to other data,
promising to facilitate data integration, exposure, sharing, and connecting.
2.5 Wiki-based integration
A weakness common to all the above approaches is that the quantity of users participations
in the process is inadequate. With the increasing volume of biological data, data integration
inevitably will require a large number of users participations. A successful example that
harnesses collective intelligence for data aggregation and knowledge collection is
Wikipedia, an online encyclopedia (http://www.wikipedia.org) that allows any user to
create and edit content. Wikipedia features collaborative integration, continuous and
frequent update, up-to-date content, huge content coverage and low cost for maintenance


48
(McLean, et al., 2007). Although there are fears of inconsistency and inaccuracy since users
can freely and anonymously change any content and/or add new content in the wiki (Arita,
2009; Bidartondo, 2008), it is testified that Wikipedia outperforms the traditional
Encyclopedia in accuracy (Giles, 2005).
In consideration of the success of Wikipedia, a wiki-based approach has been on the horizon
to store, manage and organize biological data (Giles, 2007; Salzberg, 2007; Waldrop, 2008;
Yager, 2006). The wiki-based integration makes full use of collective intelligence and efforts
for biological data integration. Representative examples include: WikiGenes (a wiki system
that combines gene annotation with explicit authorship; Hoffmann, 2008), WikiProteins (a
wiki-based system for protein annotation; Mons, et al., 2008), BOWiki (a ontology-based
wiki for data annotation and knowledge integration; Hoehndorf, et al., 2009), Gene Wiki (a
wiki for human gene annotation; Huss, et al., 2010; Huss, et al., 2008) and PDBWiki (a
scientific wiki for the community annotation of protein structures; Stehr, et al., 2010).
However, the wiki-based integration has its own shortcomings, including the unstructured
data generated, the lack of a standard format for data exchange, the lack of credit for
authorship and vulnerability to malicious editing (Lee, 2008; Potthast, et al., 2008).
3. Challenges ahead
Although a number of current efforts have been devoted to data integration, none of them
have achieved a pre-eminent impact on their field yet. Since NGS data are growing at an
exponential rate, the need for data integration is continually demanding and challenges for
data integration are greatly increasing.
3.1 Data as a service
The low-cost and high-throughput NGS technologies can generate huge amounts of data at
a relatively short period. To keep pace with the revolution of sequencing technologies,
genome sequencing projects have transitioned from classical model organisms (e.g., fly,
mouse, yeast), to other organisms (e.g., camel, dog, panda) and eventually, to sequencing
individuals within populations, exemplified by the 1000 Genomes Projecta collection of
the genomes of 1,000 humans (http://www.1000genomes.org) and the Genome 10K
Projecta genomic zoo of genome sequences of 10,000 vertebrate species
(http://www.genome10k.org). The era of $1000 personal genome sequencing is
approaching within the following years and would produce unparalleled large-scale data,
presenting considerable challenges for data integration.
It is infeasible to integrate such large amounts of data into a single point (such as a data
warehouse). Data sources are developed for different purposes and fulfill different
functions. Therefore, it is promising to establish an efficient way for data exchange among
these distributed and heterogeneous data sources. However, a dozen of data sources are
designed merely for data storage, but not for data exchange. The growing volume of
biological data also requires computer-readable approaches for data integration. To ease
data integration, data sources need to turn into service providers. In other words, data
sources should not only serve as data providers that provide data for human reading with
web interfaces (e.g., HTML), but also function as service providers that provide data for
computer interoperability via WS. Service providers supply data as a WS, facilitating
computer-to-computer interactions and thus enabling automated data integration from
multiple data sources (Hansen, et al., 2003). As mentioned, there are several different


49
protocols that can be used for creating WS. Among them, SOAP and REST have been widely
adopted (Figure 1). SOAP is a well-defined standard with XML-structured messaging for
request and response, whereas REST is relatively lightweight, relying on HTTP methods
(viz., POST, GET, PUT or DELETE). Most commercial applications expose their services as
RESTful Web APIs (Figure 1), largely due to its simplicity and easy implementation.

Fig. 1. Statistics of Web API protocols (obtained from
http://www.programmableweb.com/apis, which collects more than 3,000 Web APIs; last
access: February 27, 2011).
3.2 Standards for biological data
Due to the complex nature of biology, there are a wide variety of biological data types, e.g.,
sequence data, gene expression data, protein-protein interaction data, pathway data
(Karasavvas, et al., 2004). Data sources store different data types as different formats (Li,
2006): flat file (e.g., tab-delimited file), sequence file (e.g., FASTA), structure file (e.g., PSF
Protein Structure File), and XML file (e.g., KGMLKEGG Markup Language for describing
graph objects). Data sources often adopt their preferable data formats; even for a same data
type, data formats in different sources are often incompatible. It is also noted that new data
formats are often invented along with the development of related technologies. Examples of
newly invented file formats include SAM (Sequence Alignment/MAP; a generic nucleotide
alignment format that describes the alignment of query sequences or sequencing reads to a
reference sequence or assembly; Li, et al., 2009), and GVF (Genome Variation Format; a
simple tab-delimited format for describing genome variation data; Reese, et al., 2010). In
addition, data sources output their data in diverse formats, such as HTML, raw file formats,
and XML-based file formats. Taken together, diverse and heterogeneous data formats
complicate data exchange, posing challenges for data integration.
Standards for biological data formats can ease data exchange and integration. There has
been a successful attempt for standardizing biological pathway data. Pathway-related data
sources differed in their data representation, making data integration difficult and
inefficient. For this reason, BioPAX (Demir, et al., 2010) has been developed to deliver a
compatible standard, facilitating integration, exchange, visualization and analysis of
biological pathway data. Another effort related to cope with data incompatibilities of
bioinformatics repositories has been devoted to the standardization issues of data exchange
formats and WS (Katayama, et al., 2010). In short, establishing standard formats for
biological data can realize efficient data exchange and integration. In return, standard data
formats facilitate subsequent data analysis and visualization as well as downstream
software development.


50
Equally important, data integration also requires standardizing nomenclature and
ontologies for biological data (Rubin, et al., 2008). Suppose two data sources need to
exchange gene annotations. They must share a standard regarding gene name. Otherwise,
any ambiguity or inconsistency in nomenclature would bring a burden to data integration.
Attention has been paid to standardizing nomenclature and ontologies for biological data,
e.g., BioPortal (Noy, et al., 2009; Rubin, et al., 2006) for integrating and sharing biomedical
ontologies in National Center for Biomedical Ontology, GO (Ashburner, et al., 2000) for
standardizing the representation of gene and gene product attributes, HGNC (Seal, et al.,
2011) for standardizing human gene symbols and names, OBO (Open Biomedical
Ontologies) (Smith, et al., 2007) for creating a suite of orthogonal interoperable reference
ontologies in the biomedical domain. However, a centralized system for nomenclature and
ontologies standardization may not keep good pace with the rapid accumulation of
biological data and any gap in standardization would provoke difficulties for data
integration. A wiki-based system might be promising to harness all communities efforts in
standardizing nomenclature and ontologies collaboratively and efficiently.
3.3 WS-based pipelines
The goal of data integration is to enable combining information from different resources in
an automated fashion without human intervention, so as to handle the increasing
accumulation of biological data (Sarkar, et al., 2008). Towards this goal, data to be integrated
should be re-defined in a broader manner, which include not merely sequences and other
raw data, but also methods, tools, algorithms, analyzed results, discovered knowledge (see a
paper for knowledge integration; Clark, 2007) and even connections among people (Zhang,
et al., 2009). All kinds of data can be provided as a service. That is, raw data should be
accessible via WS, methods, tools, and algorithms that are used to analyze data should be
offered as WS (that is SaaS, Software as a Service), and analyzed results and discovered
knowledge should be also delivered as WS (Zhang, et al., 2009). As a result, WS perform a
variety of data manipulation, including data retrieval, integration, analysis, visualization,
and sharing.
A pipeline with a combination of multiple WS can achieve data integration (Zhang, et al.,
2009). Such WS-based pipelines lower technological entrance barriers and provide users
with a lightweight programming environment. WS-based pipelines feature computer-to-
computer data exchange, simplify data integration and analysis, maximize the scope of
sharing and reuse, and function as a medium to link users located anywhere with similar
research interests, and finally to form a scientific social community (SSC). SSC reflects
several key elements of Web 2.0 and enables data integration, analysis and sharing with
greater convenience, speed and efficiency (Zhang, et al., 2009). Any user may easily create
WS-based pipelines (adding value), publish them online, and subscribe to pipelines created
by other users. Consequently, pipelines may be widely shared, re-used and even integrated
into other pipelines. As a result, communications and collaborations among users in SSC can
be greatly increased, making knowledge discovery through collective intelligence possible.
In addition, SSC can also serve as a registry for collecting WS (Bhagat, et al., 2010; Pettifer, et
al., 2010).
3.4 Semantic Web Services
The ever-evolving next-generation Web (NGW), characterized as the Semantic Web, aims to
provide information not only for human, but also for computers to semantically process


51
large-scale data and automatically discover knowledge. From this view, the Semantic Web
befits well with the exponential growth of biological data and promises in providing
solutions for data integration and advancing translational research (Ruttenberg, et al., 2007).
Semantic Web technologies have been applied for data integration as mentioned above.
Nevertheless, these applications in essence belong to semantic warehouses and still have
pains for integrating dynamic data. One potential solution is to combine WS with Semantic
Web technologies and to provide Semantic WS (Matos, et al., 2010; Vandervalk, et al., 2009),
namely, RDF-based WS for automated data processing and reasoning. As mentioned, WS
are designed not only to perform a query, but also to conduct a computation. Considering
that NGS technologies can swiftly generate hundreds of gigabases of sequencing data, WS
would become increasingly data-intensive and computation-intensive (e.g., alignment of
multiple large-scale sequences). Therefore, to deal with such large-scale data management
and analysis, Semantic WS necessitate to adopt advances in high performance computing
(Schadt, et al., 2010), such as, cloud/grid computing (Bateman and Wood, 2009; Stein, 2010)
and Service-Oriented Computing (Papazoglou, et al., 2008). In addition, a Semantic WS
framework (Wilkinson, et al., 2010) is also needed, in order to set up Semantic WS
workflows or pipelines.
4. Conclusions
As a critical topic in bioinformatics, data integration bears fundamental significance for
biological studies. Efforts have been devoted to this topic and the corresponding approaches
for data integration have moved from traditional ones, e.g., data warehousing and federated
databasing, to modern ones based on several advanced technologies, e.g., Web Service,
Semantic Web and Wiki. The rapid development of sequencing technologies poses
tremendous challenges for data integration. Integration of large-scale data not only requires
adoption of informatics advances, but also needs communications and collaborations among
people in related biological communities to maximize data openness via WS, set up
standards for biological data, create Semantic WS-based pipelines and form a scientific
social community. Such community harnesses collective intelligence and collaborative
efforts for data integration, analysis and sharing, having the potential to be an ideal
community of the people, by the people, and for the people.
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Roland Kienast and Christian Baumgartner
Institute of Electrical, Electronic and Bioengineering
University for Health Sciences, Medical Informatics and Technology
Austria
1. Introduction
Contemporary life sciences research requires an understanding of systems across wide ranges
of scale and distribution. Therefore, there is an urgent need to integrate biomedical knowledge
generated by different communities and separate subelds (Shadbolt et al., 2006). Scientic
publications and curated databases together hold a vast amount of this useable knowledge.
Additionally the number, size, and complexity of life science databases continues to grow
(Kei-Hoi et al., 2009). Therefore scientists in the eld of genomics, proteomics, metabolomics,
clinical medicine and drug discovery need a concept to integrate their data, (Shadbolt et al.,
2006) which is a prominent problem (Kei-Hoi et al., 2009). But to generate such a uniform
data integration concept there are still some challenges to overcome such as handling the
variety and amount of available data, inconsistency with data heterogeneity fromthe different
sources, the autonomy and differing capabilities of the sources and a lack of standards for such
an integration concept. Many heterogeneity conicts remain in data integration due to the
lack of semantics (Gagnon, 2007). In order, to efciently exploit the knowledge from different
resources, it will be important to connect the sources in a manner that machine processes can
traverse and intelligently identify these links (Neumann et al., 2004). A promising approach
to integrate heterogeneous data sources could be the use of Semantic Web technologies. They
provide a framework to deal with the afore mentioned problems and full the requirements
for machine processing.
This book chapter provides an overview of data integration on biomedical data using
Semantic Web technologies including existing techniques (standards, specications and
methods), challenges, approaches and projects.
2. Basics of data integration
Data integration is the task of combining the data residing at different sources, and providing
the user with a unied view of the data (Cal et al., 2001; 2003). But to accomplish the task of
combining different heterogeneous sources there are some challenges to be overcome.
2.1 Challenges in integrating information from heterogeneous data sources
In the dictionary
1
heterogeneity is dened as the quality of being diverse and not comparable in
kind. In computer science this inability to compare can be divided into four different classes
(Ouksel & Sheth, 1999):
1
Websters Online Dictionary http://www.websters-online-dictionary.org

Semantic Data Integration on Biomedical Data
Using Semantic Web Technologies
3
2 Will-be-set-by-IN-TECH
System heterogeneity is a result of different hardware platforms and operation systems.
Syntactic heterogeneity is a difference of data representation formats.
Structural heterogeneity rises from different data models or structure in various data
sources.
Semantic heterogeneity results from differences in the interpretation of the meaning of
different resources.
This heterogeneity leads to some challenges in integrating information from multiple data
sources. Some general problems are (Cheung et al., 2007):
Locating Resources: To be able to integrate data it is important to nd relevant and
inter-operable data sources. But to nd such sources it is benecial to have a widely
accepted standard for describing the content of data.
Different data formats: Different resources often provide heterogeneous data formats. For
example:
structured data: e.g. different databases
semi-structured data: e.g. HTML, XML data
unstructured data: e.g text documents, images
Identify Synonyms and Homonyms: Before large scale databases where created,
researchers independently named biological entities. As a consequence many synonyms
exist. The ability to distinguish between synonyms and homonyms is very important for
data integration.
Detect Ambiguity: Different terms can be used to represent different concepts. For
example the term insulin can represent the concept hormone or drug.
Recognize Granularity: Different biological data sources may provide knowledge at
different levels of granularity. For example one source provides information about
different genetic diseases and their symptoms. Another source might only contain detailed
information about haemophilia
2
.
Scaling conicts: These conicts occur when different reference systems are used to
measure a value e.g., different date formats or size measures.
2.2 Different integration approaches
There are different approaches to integrate different data sources by using warehousing,
mediation or a combination of both.
Warehouse integration consists in catalouging the data from multiple sources into a local
database called the warehouse. All queries are executed on the data contained in the
warehouse (Hernandez & Kambhampati, 2004; Kugler et al., 2008; Pfeifer et al., 2007).
The task of importing data from a source into the warehouse is called the ETL (Extract -
Transform - Load) process.
Advantages: Warehousing eliminates various problems such as network bottlenecks,
low response times, and temporarily unavailable sources. It allows to lter, validate,
modify, and annotate the data obtained from the sources (Davidson et al., 1995).
2
Haemophilia is a genetic disease which interferes with blood clotting.
58 Bioinformatics Trends and Methodologies
Semantic Data Integration on Biomedical Data using Semantic Web Technologies 3
Disadvantages: It is necessary to build and maintain the warehouse and there is a
danger of antiquated data. Therefore the warehouse system must regularly check the
underlying sources for new or updated data and modify the local copy of the data if
required (Davidson et al., 1995).
Mediator based integration concentrates on query translation. Amediator is a systemwhich
provides a query translation from a single mediated schema to the local schema of the
underlying data source (Hernandez & Kambhampati, 2004). The data ow between
mediators and data sources is provided by software components called Wrappers. Unlike
warehousing, data is not centrally stored but it is accessed directly from the distributed
sources.
Advantages: The data is always up to date and there is no need to maintain a storage
system.
Disadvantages: Mediator based integration is sensitive to network bottlenecks, low
response times and temporarily unavailable sources.
An other possibility is using Semantic Web technologies. The goal of the Semantic Web
approach to data integration is to add machine readable metadata to resources and to dene
and describe relations among them. This makes it easier to automatically process and
integrate information available within the different resources (W3C, 2004a) (see gure 1).
Different
databases
Different
articles
Different
websites
Different
databases
Different
articles
Different
websites
Data (RDF, RDFS)
Query (SPARQL)
Ontologies (OWL)
Semantic Web TechnoIogies
Fig. 1. The goal of data integration using Semantic Web technologies. Right: The user must
consult several resources individually through different user interfaces to derive a result.
Left: Semantic Web technology allows the integration of various heterogeneous resources.
The system can process the data and provide the results to the user.
3. Semantic Web in a nutshell
Tim Berners-Lee , the director of the World Wide Web Consortium (W3C), coined the term
Semantic Web (Berners-Lee et al., 2001) and it is mainly used to describe the model and
technologies provided by the W3C which is the main international standards organization for
the World Wide Web. The aim of the Semantic Web is to add structured meta-information to
59 Semantic Data Integration on Biomedical Data Using Semantic Web Technologies
existing documents and data in order to give it a well dened semantic meaning. This enables
machines to process semantic information but not human speech and writings (Berners-Lee
et al., 2001). This semantic extension makes it easier for machines to automatically process
and integrate information available on the Web (W3C, 2004a).
The basic idea behind the Semantic Web is to add machine readable metadata
3
to resources
within the World Wide Web to dene and describe relations among them. Semantic Web
technologies are able to assimilate this gained information. Furthermore, they do not build a
separate web, but function as an extension of the current web. The Semantic Web technology
consists of a hierarchical use of various standards and technology in which each layer uses the
capabilities of the layers below. The architecture of the semantic web is illustrated in gure 2.
A brief description of each layer is summarized below:
User interface and applications
Identifiers: URI
Character set:
UNICODE
Syntax: XML
Data interchange: RDF
Taxonomies: RDFS
Rules: RIF
Ontology:
OWL Query:
SPARQL
Unifying Logic
Proof
Trust
C
r
y
p
t
o
g
r
a
p
h
y
Fig. 2. Semantic Web stack
Character Set: UNICODE denes a fundamental coding standard for data.
Identiers: URI is a standard for the identication of resources.
Syntax: XML provides a fundamental syntax for structured documents.
Data interchange: RDF is a data model for resources and relations between them. It uses
the XML syntax.
Taxonomies: RDFS is an extension of RDF and provides a vocabulary for describing RDF
resources.
Rules: RIF denes the rules of semantic data.
Ontologies: OWL offers more opportunities to add semantic information to resources than
RDFS.
Query: SPARQL is a protocol and a query language for RDF.
Unifying Logic allows to draw a conclusion.
Proof attempts to verify the conclusions.
Trust provides trusted principles and authentication methods between different agents.
3
According to the Dictionary of Computing (http://dictionary.reference.com/browse/
meta-data) metadata is denitional data that provides information about or documentation of other data
managed within an application or environment. In relation to the Semantic Web Tim Berners-Lee denes
metadata as (Berners-Lee, 1997): machine understandable information about web resources or other thing.
In short, metadata is data about data.
4. Semantic Web approach to data integration
The W3C denes the abilities of the Semantic Web as follows (W3C, 2011):
The Semantic Web is about two things. It is about common formats for integration and combination
of data drawn from diverse sources, where on the original Web mainly concentrated on the interchange
of documents. It is also about language for recording how the data relates to real world objects. That
allows a person, or a machine, to start off in one database, and then move through an unending set of
databases which are connected not by wires but by being about the same thing.
Semantic Web approach to data integration can deal with heterogeneity by providing
structured meta-information to existing documents and data. A key feature integrating
information is the use of semantics which gives meaning to a word or concept (Gardner, 2005).
Semantics can solve the problem of homonyms and synonyms between different sources
because it is able to ensure the equivalence of two concepts which might have different names
and forms (synonyms) or the dissimilarity of two concepts which might have the same name
and form (homonyms). Semantics describe relationships between concepts. This enables a
fully descriptive representation of the available information, showing the interaction between
concepts and allows inferences. Semantic Web technologies provide a tool to describe such
semantic: The use of Ontologies. In order to achieve a benecial use of ontologies, it is
important to link the data to its semantic knowledge. In other words, it is important to
annotate instances to ontologies. But these data often have different data formats (relational
databases, text les, web sites, etc.). Adding metadata can solve this problem. But to benet
from this metadata, it should be standardized and machine readable. Such a kind of metadata
provided by the Semantic Web technology is based on the Extensible Markup Language
(XML).
4.1 Important technologies for data integration in greater detail
This section describes the most important technologies which are needed for a semantic data
integration based on Semantic Web technologies.
4.1.1 URI (Uniform Resource Identiers)
A URI is dened in RFC3986 (Berners-Lee et al., 2005): A Uniform Resource Identier (URI) is
a compact sequence of characters that identies an abstract or physical resource. In the web URIs
typically refer to websites or other data. But in general URIs can be used to generate unique
identiers for different resources. For example the namespaces of a XML (Extensible Markup
Language) document are identied by URI references. Also, in RDF (Resource Description
Framework), URIs are used to refer to resources (Hitzler et al., 2008).
4.1.2 XML (eXtensible Markup Language)
XML is a machine readable, standardized meta-language. It is an important basic technology
for the Semantic Web (W3C, 2001) with which it is possible to create structured documents.
These documents are text based and provide their data in a hierarchical and logically
structured form which can be read by humans and by machines. It is an markup based
language and uses tags for this purpose. In Informatics markup languages are used to extend
parts of an document with additional information to describe it in more detail. This additional
information is also called metadata.
Problems with XML and data integration:
XML is standardized, machine readable and denes the syntactical structure of a document.
But in the view of the Semantic Web, XML tags are not much better than the natural language
(Hitzler et al., 2008). These tags can be ambiguous, their relationship is not clearly dened
and they provide no meaning for machines.
4.1.3 RDF (Resource Description Framework)
Originally RDF was designed for adding metadata to web resources but it has become a
framework for adding semantic information to resources. RDF is machine readable. Therefore
it enables the encoding, exchange, and reuse of structured metadata and allows structured
and semi-structured data to be mixed, exposed and shared across different applications(W3C,
2010a) which can make use of the semantic information (Fensel, 2004).
RDF provides a simple data model for describing relationships between resources in terms
of named properties and their values. While XML can only describe documents in a tree
structure, RDF is a framework for representing information about resources in the form of a
directed graph. An edge of this graph describes the relationship between two resources. RDF
documents can be written in Notation 3 (N3) (W3C, 2005), N-Triples (W3C, 2004d), Turtle
(W3C, 2008c) syntax or in a XML syntax. This XML syntax is called RDF/XML (W3C, 2004f).
But XML can only describe a tree structure whereas RDF represents a graph. Therefore it
is necessary to serialize these complex data objects into strings. RDF uses so-called triples
(3-tuples) to describe relationships between resources to serialize the graph. A RDF-triple
consists of only three elements (W3C, 2004g):
1. The subject: Is a RDF URI reference or a blank node.
2. The predicate: Is a RDF URI reference.
3. The object: Is a RDF URI reference, a blank node or a literal.
A triple is conventionally written in the order subject, predicate, object and can be illustrated
by a node and directed arc diagram (see gure 3). A set of these triples form a directed graph.
Aproblemin RDF is that URI references can not describe a conclusive semantic interpretation
subject object predicate
Fig. 3. Illustration of a triple
of RDF coded information (Hitzler et al., 2008) because a URI can also be a homonym or
synonym of another URI. This principle is also known as Non Unique Name Assumption. A
solution to this problem is to use thematic vocabularies such as FOAF (Friend of a Friend)
vocabulary which can be used for linking people and information about them (Brickley &
Miller, 2010).
4.1.4 Ontologies to share semantic information
(Gruber, 1993) denes an ontology as: An ontology is an explicit specication of a
conceptualization. This denition was slightly modied by (Studer et al., 1998): An ontology
is a formal, explicit specication of a shared conceptualization.
A conceptualization refers to an abstract model of a phenomenon in the world which identies
the relevant concepts of that phenomenon. Explicit correlates to the formed types of concepts
and their limitations, which are dened explicitly. Formal is based on the fact that an
ontology should be machine readable. Shared means that an ontology should cover matching
knowledge. This knowledge is not limited to an individual and is accepted by a group (Fensel,
2004; Studer et al., 1998).
This abstract denition is understandable on the basis of a simple example. It contains a brief
abstract of the ontology of animals (see gure 4).
The abstract model includes the terms animal, sh, mammal and puma. These terms come from
animal
fish mammal
puma
Fig. 4. A brief abstract of the ontology of animals.
the phenomenon of animals. Every term is explicit. The term puma is explicitly dened
as a animal. It cannot be confused with the clothing brand Puma. Puma also have clear
limitations: a puma is an organism which is a mammal and not a sh and belongs to the
animals. An ontology is represented as a directed graph. A graph is formal and machine
readable. The ontology is also shared because not only one individual can infer knowledge
and it is accepted by a group of biologists.
The structure of an ontology is a directed acyclic graph. That makes it possible to support
complex relationships which allow terms to have more than one parent. For example the
Gene Ontology
4
term GO:0070229 : negative regulation of lymphocyte apoptosis is a subclass of
GO:2000107 : negative regulation of leukocyte apoptosis and GO:0070228 : regulation of lymphocyte
apoptosis. Ontologies are able to describe the semantic of the information sources in order to
make their content explicit. A basic module of ontologies is the so called triple. Broadly
dened, a triple contains two terms and a relation between them
5
. With these elements an
ontology can be represented as a directed graph. The terms are the nodes and the relations are
the edges of the graph (Smith et al., 2005).
4.1.5 RDFS (RDF Schema)
Like XML, RDF only provides a syntax for exchanging data. RDF properties can be considered
as attributes of resources and also represent relationships between them. But it provides
no mechanisms for adding a vocabulary to describing these attributes or relationships.
RDFS, or also called RDF Vocabulary Description Language, extends RDF to describe such
vocabularies (W3C, 2004c;e) and add terminological knowledge (schema knowledge) to this
vocabulary. For that reason it can be seen as a semantic extension of RDF. RDF Schema
vocabulary descriptions are written in RDF syntax (W3C, 2004e). It makes statements
about the semantic relationship between terms within an arbitrarily dened vocabulary
inside a RDFS document. This ability to dene terminological knowledge allows RDFS to
create light-weight ontologies (Hitzler et al., 2008; Volz et al., 2003) to describe semantic
dependences within a domain.
Figure 4 shows a simple RDFS document in graph representation. RDFS organize RDF
statements hierarchically into classes (terminological knowledge) and instances (assertional
knowledge). Properties are used to describe relationships between classes. The terminological
part includes the ontology while the assertional part presents conclusions about concrete
4
see section 5.3.2
5
see section 4.1.3 for a detailed description
qualities of the subject. This ontology describes, for example, that the class cell is a subclass
of the class organ and that every cell consumes energy. Further, it is possible to derive
implicit knowledge. If the muscle cell is an instance of the class cell and the ATP (Adenosine
Tri-Phosphate) is an instance of high energy chemical bond, then it is possible to infer that a
muscle cell is part of a human and ATP is a kind of energy.
ex:Organ
ex:Human
ex:Cell
ex:high-
energy_chemical_bond
ex:Energy
ex:consumes_energy
ex:consumes
rdfs:subPropertyOf
rdfs:subClassOf
rdfs:subClassOf
rdfs:subClassOf
rdfs:domain
rdfs:range
muscle cell ATP ex:consumes_enery
rdf:type rdf:type
t
e
r
m
i
n
o
l
o
g
i
c
a
l
k
n
o
w
l
e
d
g
e
(
R
D
F
S
)
a
s
s
e
r
t
i
o
n
a
l
k
n
o
w
l
e
d
g
e
(
R
D
F
)
(subject)
(predicate)
(object)
Class
Property
Instance
Fig. 5. Simple RDFS-Ontology in graph representation
4.1.6 OWL (Web Ontology Language)
OWL is designed to enable machine processing of information content. OWL can explicitly
represent the meaning in terms of vocabularies and their relationship with each other to build
an ontology. Since October 2009 the version OWL 2 is recommended from W3C(W3C, 2009a).
In contrast to RDFS, OWL has more opportunities to expressing meaning and semantics.
Therefore OWL can be seen as an extension of RDFS (W3C, 2004b). An OWL ontology is
an RDF graph which consists as a set of triples. It also can be written in different syntactic
forms but the most common syntax is RDF/XML for representing these triples.
OWL provides three increasingly expressive sub-languages (Alesso & Smith, 2006):
1. OWL Lite to generate a classication hierarchy and simplify constraints. -> Easily
implementable
2. OWL DL (description logic) supports maximum expressiveness while retaining
computational completeness and decidability. -> Mechanizable logic
3. OWL Full provides maximum expression and syntactic freedom of RDF but with no
computational guarantees. -> Complete Logic
Since OWL is an extension of RDFS and therefore also from RDF, any RDF document
will generally be in OWL Full. OWL DL and OWL Lite also extend the RDF vocabulary,
but they put restrictions on the use of this vocabulary (W3C, 2004a;b) for better machine
processing. These restrictions guarantee computational completeness and decidability of
reasoning systems like FaCT++ (Tsarkov & Horrocks, 2006) and the Pellet (Sirin et al., 2007)
which are able to reason over OWL 2 ontologies (Grau et al., 2008). This is achieved because
OWL Lite and DL are basically very expressive description logics (DL) where OWL DL is
based on the SHOIN(D)DL (Hitzler et al., 2008) and OWL Lite to the slightly simpler
SHIF(D)DL.
Description Logics (DL) stem from semantic networks (Donini et al., 1996). They model
concepts (equal to a class in OWL), roles (equal to a property in OWL) and individuals (equal
to a object in OWL), and their relationships. Therefore they can be used to represent the
knowledge of a specic domain in a formal and structured way. Here the context of ontologies
is clearly visible. As described in 4.1.4 an ontology consists of axioms, which are used
to provide information about classes and properties of a specic domain. The knowledge
which is provided by DL is divided into a TBox and an ABox (Donini et al., 1996). The
TBox (terminological box) contains sentences describing concept hierarchies and the ABox
(assertional box) contains sentences about the individuals and where they are in the hierarchy
(Van Harmelen et al., 2008). For example the statement Every protein is made of amino acids
belongs to the TBox, while the statement Leucine is a amino acid belongs to the ABox.
The drawing of logical conclusions in OWL are based on the concept of the so-called Open
World Assumption (OWA). In contrast to the Closed World Assumption (CWA), this assumption
species that statements are neither true nor false if they can not be derived from a set of facts
based on inference rules. The OWA does not assume that a answer is false unless it can be
absolutely proven that the answer is false (Pollock, 2009). Listing 1 shows an example of both
assumptions.
Listing 1. Example for the open- and closed world assumption
Knowledge Base: The protein p53 is involved in apoptosis.
Query: Is the protein p53 involved in cell repair?
Answer: CWA: No.
OWA: Maybe or unknown.
4.1.7 SPARQL (Simple Protocol and RDF Query Language)
SPARQL is a protocol and query language for RDF which since January 2008 is an ofcial W3C
recommendation (W3C, 2008a). SPARQL queries often contain a set of triple patterns. These
patterns, or also called basic graph patterns, look like RDF triples. The difference is that every
subject, predicate or object, can be expressed as a variable. A match can be found by replacing
variables through substituting RDF terms. If the result of the substitution is equivalent to a
subgraph of the RDF data a match is found. For example, to nd the meaning of the acronym
ATP and where it is produced, the SPARQL query would look like Listening 2.
Listing 2. Simple SPARQL query
PREFIX ex: <http://example.com/>
SELECT ?longName ?part
WHERE
{ex:ATP ex:hasLongName ?name.
?name ex:producedIn ?part}
The SELECT clause denes the variables which appear in the result and the WHERE clause
provides the basic graph pattern. In this case the graph pattern consists of two triple patterns
with two single variables.
As a simple knowledge basis following RDF data (see Listing3) in Turtle notation is used.
top
-level
ontologies
top-domain
ontologies
domain ontologies
local
ontologies
u
s
a
b
i
l
i
t
y
r
e
u
s
a
b
i
l
i
t
y
Fig. 6. Classication of ontologies. The reusability decreases with increasing specication.
The availability behaves exactly opposite.
Querying this RDF 3 data with the SPARQL query 2 obtained the result shown in table 1. It
name part
Adenosine Tri-Phosphate http://example.org/cell/mitochondrion/
Table 1. Result of SPARQL Query 2 on RDF Data 3
is also possible to generate complex graph patterns out of a number of simple patterns or to
dene lters to restrict the result. SPARQL provides four query forms which form a result
SELECT, ASK set or RDF graphs CONSTRUCT, DESCRIBE out of the pattern matching. To
serialize a result from a SELECT or from an ASK query into a XML document the SPARQL
Variable Binding Results XML Format(W3C, 2008b) can be used.
5. Using ontologies for data integration
Biomedical ontologies play an important role in the process of data integration and support
both approaches for data integration: warehousing and meditation (Bodenreider, 2008).
Ontologies are a type of controlled vocabulary that attempt to capture the knowledge
of a specic domain. This is the standardization required from warehousing approaches,
where different sources are transformed into a common format and converted to a common
vocabulary. On the other hand, the mediation-based approach ontologies can be used for dening
global schema and mapping between the global schema and local schemes of the sources to
integrate. An example of a system using this approach is ONTOFUSION (Perez-Rey et al.,
2006). The terminological part of ontologies, which contain a list of names for the entities
represented in these ontologies, is also an important resource for natural language processing
(Altman et al., 2008).
Based on their granularity, ontologies can be divided into four classes (see gure 6):
Top-level ontologies describe very general concepts which are independent of a particular
problem or domain (Guarino, 1998) and are highly reusable across specic domains.
Listing 3. Simple RDF data
PREFIX ex: <http://example.com/cell>
ex:ATP ex:hasLongName "Adenosine Tri-Phosphate"
ex:ATP ex:producedIn ex:mitochondrion
Top-domain ontologies contains core concepts of a given domain. For example: Organism
or Cell for a biological domain. They work like an interface between top-level and domain
ontologies (Stenzhorn et al., 2008).
Domain ontologies include only domain specic concepts and therefore only describe a
certain domain.
Local ontologies describe the semantic of a single information resource.
The ability of ontologies to provide a map of concepts in relationships enables semantic data
integration. In this context, ontologies are used to describe the semantics of the data sources
in order to make their content explicit (Boury-Brisset, 2003). The integration can take place
on an extremely granular level to map data from different resources, no mater if the resources
contain structured or unstructured data (Gardner, 2005).
Ontology-based approaches to data integration usually provide a three-layer architecture
where a semantic layer working as a mediator is between the presentation layer and the
physical layer. This semantic mediator exploits mapping models and transforms queries into
execution plans. Wrappers exploit the description of the data sources at the physical layer.
This enables a transparent access to diverse data sources by using a unied query language
(Boury-Brisset, 2003) like SPARQL. Ontologies are used in the mediator layer because they
provide a common vocabulary for the integration of data, where each concept has a unique
dened name, associated properties and clearly dened synonyms. Furthermore, an ontology
is not a rigid structure, it can grow with time and can be connected to other ontologies.
Wache (Wache et al., 2001) describes three approaches for ontology-based data integration:
Single ontology approach: This approach uses only a single global ontology to integrate
different sources. All information sources are related to the global ontology. The global
ontology can be a combination of different specialized ontologies. This approach requires
data sources with a similar view on the domain and a similar granularity. A disadvantage
of this approach is that the integration of new information sources can lead to big changes
in the used ontology.
Multiple ontologies approach: The semantic of an source is described by its own local
ontology. There is no common vocabulary and therefore inter-ontology mapping is
required. An advantage of this approach is that new data sources, and their local
ontologies, can be easily integrated. But the lack of common vocabulary can make the
mapping between ontologies very difcult to dene.
Hybrid approach: This is a combination of the two preceding approaches. As with the
multiple ontologies approach, resources are also described by local ontologies. But to
avoid the disadvantages and to make these ontologies comparable, they are built from
a shared global vocabulary. This vocabulary contains basic terms of a domain and allows
querying through a shared vocabulary. The vocabulary can also be an ontology. Then it is
also possible to dispense with the mapping between the local ontologies and only dene
mappings between the shared global ontology and the local ones. New sources can be
easily added with no need to modify existing mappings.
An example of using ontologies for data integration in biomedicine is the Gene Ontology
Annotation (GOA)
6
project run by the European Bioinformatics Institute (EBI). GOA is based
on the single ontology approach and has as target to provide high quality electronic and
manual annotations to the UniProt knowledgebase
7
(UniProtKB)(Barrell et al., 2009). For
6
http://www.ebi.ac.uk/GOA
7
http://www.ebi.ac.uk/uniprot
this purpose, GOAuses the standardized vocabulary of the Gene Ontology (GO) 5.3.2 and the
International Protein Index (IPI) (Camon et al., 2004). The IPI offers complete, non redundant
data sets representing the human, mouse and rat proteomes (Kersey et al., 2004).
Another advantageous feature of ontologies is that terms are organized in a hierarchical
manner (Stein, 2003). That means more specic terms are specializations of more general
terms. This could help to nd the most specic common term shared by two data sources. An
example of such a benet could look like the following:
One research group might create a database in which gene products annotated to the negative
regulation of T cell apoptosis-class of the Gene Ontology. Another group might identify gene
products which negatively regulate the programmed cell death. If both groups use the terms
of the GO, the two databases can be integrated by nding the most specic common term by
traversing up the hierarchy (see gure 7). Without such an organized hierarchy of common
concepts, the integration task comes down to tedious and error-prone work by hand (Stein,
2003).
Source A
Source B
t
r
a
v
e
r
s
i
n
g

u
p
most specific common term
Fig. 7. Find the most specic common term by traversing up the hierarchy.
(This gure shows an extract of the Gene Ontology http://www.geneontology.org)
5.1 Examples of existing top-level ontologies
5.1.1 Descriptive Ontology for Linguistic and Cognitive Engineering (DOLCE)
DOLCE is the rst module of the WonderWeb
8
foundational ontologies library. It aims
at capturing the ontological categories underlying natural language and human commonsense.
(Masolo et al., 2003). The Dolce foundational ontology and its extensions provide a
domain-independent framework to build ontologies on the basis of highly-reusable patterns.
5.1.2 Basic Formal Ontology (BFO)
The BFO is narrowly focused on the task of providing a genuine upper ontology which can
be used in support of domain ontologies developed for scientic research, for example in
biomedicine within the framework of the OBO Foundry (IFOMIS, Saarland University, 2010).
5.2 Examples of existing top-domain ontologies
5.2.1 The Unied Medical Language System (UMLS)
Having identied terminology is a key factor for data integration (Bodenreider, 2004)
therefore the UMLS was developed by the National Library of Medicine (NLM)
9
and consists
of three knowledge Sources which can be used separately or together (U.S. National Library
of Medicine, 2010):
Lexical resources: SPECIALIST lexicon: Intends to be a general English lexicon which
includes many biomedical terms.
8
http://wonderweb.semanticweb.org
9
http://www.nlm.nih.gov
Terminological resources: Metathesaurus: Includes biomedical and health related source
vocabularies, concepts and the relationships between them.
Ontological resources: Semantic Network: Contains categorization of all concepts
represented in the UMLS Metathesaurus and relationships between these categories.
The Semantic Network (SN) can be seen as a collection of ontologies. In order to use these
with Semantic Web technologies it is necessary to convert the SN to OWL DL. There are
some approaches to map or convert UMLS SN to RDF (Zeng & Bodenreider, 2007), to OWL
(Kashyap & Borgida, 2003; Schulz et al., 2009) or only parts to OWL (Chabalier et al., 2007).
But there are formalism problems concerning this task like the complex semantics or the rich
attribute set of the UMLS SN.
5.2.2 BioTop
BioTop is a top-domain ontology for the Life Sciences with the goal to provide an ontologically
sound layer for linking and integrating various specic domain ontologies from the life sciences
domain. (Beisswanger et al., 2008).
5.3 Examples of existing domain ontologies
5.3.1 Open Biological and Biomedical Ontologies (OBO)
The OBOFoundry is a collaborative experiment involving science based ontology developers.
The goal is to create orthogonal inter-operable reference ontologies in the biomedical domain
(OBO Foundry, n.d.). These ontologies typically have the OBO at le format. Like OWL,
OBO is also an ontology representation languag (Richter, 2006). Ontologies based on the OBO
at le format can be bi-directionally converted to the OWL-DL format (Aranguren et al.,
2007; Hoehndorf et al., 2010; Smith B. et al., 2007). The two most signicant OBO are the Gene
Ontology (GO), which contains the principle attributes of gene products, and the Sequence
Ontology, which describes the features of biological sequences.
5.3.2 Gene Ontology (GO)
The GO project
10
contains dened terms which represent gene product properties. The GO
covers three aspects of separate ontologies(Gene Ontology, n.d.):
Molecular function: the elemental activities of a gene product at the molecular level, such
as binding or catalysis.
Biological process: operations or sets of molecular events with a dened beginning and
end, pertinent to the functioning of integrated living units: cells, tissues, organs and
organisms.
Cellular component: the parts of a cell or its extracellular environment.
5.3.3 Sequence Ontology (SO)
The SO Project
11
contains dened terms which describe the features and properties of
biological sequences. SO is a sister project of the GO and also part of OBO (Eilbeck et al.,
2005).
10
http://www.geneontology.org
11
http://www.sequenceontology.org
6. Relational database integration using the Semantic Web Approach
A lot of biomedical data is available to the scientic community on the web. Much of this
information is stored in a variety of different databases. The content of these databases differ
from the type of biological data they provide (Baker & Cheung, 2007). For example:
Sequence databases like EMBL Nucleotide Sequence Database (EBI, n.d.a) or NCBIs
GenBank (NCBI, 2004).
Microarray gene expression databases like the EMBL ArrayExpress Archive (EMBL-EBI, n.d.),
NCBIs Gene Expression Omnibus (GEO)(NCBI, n.d.) or the Stanford Microarray Database
(SMD) (Stanford University, n.d.).
Pathway databases like KEGG (Kanehisa-Laboratories, n.d.) or the Human Protein
Reference Database (HPRD) (Keshava Prasad et al., 2008).
Proteomic Databases like the UniProt (EBI, n.d.b).
Computational analyses of biological data often require using multiple datasets. Currently,
the integration of different data sets usually happens manually. This approach is very time
consuming which requires integrated datasets with rich, exible and efcient interfaces (Smith
A. et al., 2007).
6.1 Problems of heterogeneous database integration
Technical heterogeneity results from different access protocols, le formats, query
languages and so on.
Data model heterogeneity arises because of different models storing the same data.
Semantic heterogeneity occurs during combination of different databases with various
but related data. For example combine a gene database to a protein database. A gene may
have gene products and therefore these two databases are related.
Resolving such heterogeneity and enabling database integration is a key problem which
the Semantic Web aims to address (Baker & Cheung, 2007). Therefore a mapping language
between RDF and relational databases called RDB2RDF is under development.
6.2 RDB2RDF
A workshop hosted by the W3C on RDF accesses to Relational Databases in October 2007
resulted in creating a RDB2RDF Incubator Group (W3C, 2010b), which operated from 2008 to
2009. The objective of this group was to create a group to develop a standardized mapping
language between RDF and relational databases (W3C, 2009c). The resulting RDB2RDF
working group started in 2009 with: The mission of the RDB2RDF Working Group, part of the
Semantic Web Activity, is to standardize a language for mapping relational data and relational database
schemas into RDF and OWL, tentatively called the RDB2RDF Mapping Language, R2RML. (W3C,
2009b). The results of this working group are scheduled for release September 30
th
,2011.
The RDB2RDF mapping language could be used in two ways (see gure 8):
1. To extract the data from the relational database and store the content in RDF. In this
case the data is physically converted to RDF in a ETL (Extract-Transform-Load) and then
stored in a RDF triple store. An advantage of this approach is its easy implementation. A
disadvantage is that there is always a separate copy of the relational data.
2. To generate virtual mapping between the Semantic Web technologies and the relational
database. This virtual mapping queries via SPARQL which will be translated into SQL
queries on the underlying relational data.
Database A
RDF copy of
relational data
Database B
Mapping
RDF <-> relational data
ETL process
SQL query
Semantic Web
(Technologie)
Query (e.g. SPARQL)
Query (e.g. SPARQL)
Query
R
D
B
2
R
D
F
Fig. 8. Two approaches which use the RDB2RDF mapping language
7. Data integration and knowledge acquisition from biomedical literature
The quantity of biomedical literature is steadily growing with a rate of several thousand
papers per week (Ananiadou et al., 2006). A large percentage of information is encoded in
literature (Krallinger et al., 2008). But for a scientist it is next to impossible to read all relevant
literature on a specic topic. Therefore it is important to extract semantic information out of
literature to enable machine processing. This section provides an overview of how Semantic
Web technologies support this task. Ontologies in particular are able to handle this inux of
information and enable the data integration of biomedical literature (Spasic et al., 2005). Basic
techniques to extract information from natural language are text mining (TM) and natural
language processing (NLP).
Sections of TM are:
1. Information retrieval (IR): Retrieve of relevant documents.
2. Information extraction (IE): Extraction of relevant information from the document.
3. Data mining (DM): Discover of associations between information extracted by IE.
7.1 Information retrieval (IR)
The process of IR can be improved by adding a semantic layer. This layer formulates semantic
queries, offering a higher expressive power than keyword matching (Spasic et al., 2005).
However, adding semantic information to enhance the process of nding relevant information
is generally a main part of Semantic Web technology. An example of such query systems are:
GoPubMed (www.gopubmed.org): This system submits keywords to PubMed
12
. The
resulting abstracts are matched against Gene Ontology and Medical Subject Headings
(MeSH) (Doms & Schroeder, 2005) to be classied. To nd a match, a term extraction
algorithm based on local sequence alignment is used (Delfs et al., 2004). In other words
GoPubMed organize the results of a PubMed search using the GO.
12
PubMed (http://www.pubmed.gov) is a literature database provided by the National Library of
Medicine and the National Institutes of Health.
Textpresso (http://www.textpresso.org): A tool for neuroscience which has its own
literature lled database. It uses a custom ontology to query nine different categories
(Mller et al., 2008).
7.2 Information Extraction (IE), Data Mining (DM)
There are two ways to enhance the process of IE respectively, use TM and NLP supporting
literature data integration based on Semantic Web technologies:
1. Ontology assisted extraction of meta-information from literature.
2. Semi-automatic or automatic engineering of ontologies by a specic domain based on
information extracted from literature.
Generally, text mining is used to aid experts in extracting knowledge from a large volume of
text by automatically ltering relevant information. A known problem is to nd terms which
represent specic classes of biomedical entities (e.g. protein names). This process is called
Named Entity Recognition (NER). The integration of knowledge, supported by ontologies, can
improve NER. The goal is to extract terms and map them to concepts of a domain specic
ontology. A challenge in this process is the myriad variations of terms used to describe things
in natural language. Approximately one third of term occurrences are variants (Jacquemin,
2001) and therefore only synonyms of known terms. Another problem is the specic
terminology in biomedical texts. To have terminological knowledge is of vital importance
to TM for characterizing knowledge in the domain. This knowledge is stored in ontologies
and can enhance the process of IE by (Spasic et al., 2005):
Using Ontology as a training set for NER by reducing it to a list of classied terms. This
can be done in two ways:
Passive ontology use (Ontology-based IE): The goal of this approach is to map recognized
terms in ontology concepts by look-up.
Active ontology use(Ontology-driven IE): involves ontologies directly in the process of
term recognition.
Using ontologies to improve machine learning approaches for TM tasks, such as term
classication, term clustering and term relation extraction.
7.3 Semi-automatic or automatic ontology engineering
An advanced task is semi-automatic or automatic engineering of ontologies from a specic
domain on the basis of information extracted from literature. Currently the development of
ontologies is largely a manual process, based on personal experience and intuition (Alexopoulou
et al., 2008). Two primary parts of this process are:
1. Extracting terms which represent a concept in the specic domain.
2. Finding relationships between different concepts.
For an automatic terminology development it is important to extract terms from a text. This
automatic identication of possible candidates for terms is called automatic term recognition
(ATR). At the moment ATR is not able to fully automate the process of ontology design, but
it can speed up this process by providing lists of useful domain-specic terms extracted from
domain specic literature. Therefore it can support a semi-automatic creation of ontologies
(Alexopoulou et al., 2008). Examples of frameworks which support ATR and further identify
the semantic relations between them are:
Text2Onto: This is a framework for ontology learning from textual resources. It is based on
algorithms calculating the relative term frequency (Cimiano & Volker, 2005).
OntoLearn: OntoLearn is based on a linguistic processor and a syntactic parser. It is able
to extract syntactically plausible terminological noun phrases (Navigli & Velardi, 2004;
Velardi et al., 2005).
8. Challenges in data integration using Semantic Web technologies
8.1 Uniform naming
A challenge faced by data integration is the individual naming of objects. For example a
KEGG
13
entry refers to a collection of proteins involved in a pathway whereas a UniProt
entry refers to a class of proteins, a class of variant proteins or some viral protein. To integrate
these two resources mapping is required. One approach is to designate an authoritative
names commission to manage the denitive list of such names (Stein, 2003). An example
is the HUGO Gene Nomenclature Committee
14
for gene names and symbols (short-form
abbreviation). But because of the dynamic in the eld of biomedical research this approach
rarely work in practice (Stein, 2003).
Another way could be the creation of globally unique biological identiers. For this purpose
URIs can be used which allows for the unique identifying of resources. This is central for
the use of Semantic Web technologies. Therefore a process is needed which routinely assigns
URIs to objects (Shadbolt et al., 2006) to create common, shared identities and names (Goble
& Stevens, 2008).
8.2 Extraction of the semantic information out of existing knowledge
For efcient use of Semantic Web technologies, it would be useful to automatically or
semi-automatically extract the semantic information from existing sources. Therefore a big
challenge is to develop methods which support such a task. This would aid two main tasks in
data integration using Semantic Web technologies:
1. Annotate sources to existing ontologies: This is a process which extracts information from
the data source to automatically or semi-automatically annotate this source to an existing
ontology.
2. Creation process of ontologies: This is a task which extracts information from
different data sources belonging to a specic domain. The goal is to automatically or
semi-automatically create an ontology based on the extracted domain information.
A large percentage of information encoded in literature (Krallinger et al., 2008) is in the
form of natural language. Some approaches for such semantic information extraction from
literature can be found in section 7.
8.3 Ontology development, maintenance and quality
Ontologies must be developed, managed and endorsed by committed practice communities
(Shadbolt et al., 2006). Furthermore, an ontology is a living structure which means that
concepts can change constantly because of new knowledge. They can be added, changed,
replaced or removed. Therefore ontologies are not xed for all time and must be constantly
maintained. Another problem is the quality assurance (QA) of ontologies. According to
Gruber (Gruber, 1995) design and quality criteria for ontologies should be:
13
KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/)
14
http://www.hugo-international.org/comm_genenomenclaturecommittee.php
1. Clarity: The intended meaning should be clearly dened and the denitions should be
objective.
2. Extendibility: The effort needed to extend an ontology without invalidating it.
3. Minimal encoding bias: No particular symbol-level encoding should be used to specify
terms.
4. Minimal ontological commitment: An ontology should use as few terms and relationships as
possible to describe the domain being modeled.
5. Coherence: The content of the ontology should be coherent. In other words inferences
should never contradicts a denition.
The quality of an ontology can be checked either collaboratively by users or centrally, by
experts. To test the coherence of an ontology Ontology-Reasoners like Pellet
15
could be used.
Ontology Reasoning is a process of automated logical inference of knowledge with ontologies.
It is used to check the consistency of knowledge models and to infer new knowledge in
accordance with the laws of logic.
8.4 Mapping, merging, alignment and integration of ontologies
Many individual ontologies are created and therefore the semantic mapping between different
ontologies has become a core issue for the Semantic Web and data integration using its
technology. To handle the increasing number of ontologies it is necessary to develop
semi-automatic or automatic approaches (Ehrig & Sure, 2004).
The problem with the mapping of ontologies is their heterogeneity which can be divided into
metadata heterogeneity and instance heterogeneity (Tang et al., 2006). Metadata heterogeneity is
concerned with the intended meaning of the information held in different ontologies and deal
with structural conicts and name conicts. Structural conicts arise from ontologies which
cover the same domain but have different taxonomies (Ehrig & Sure, 2004), and naming
conicts concern homonyms and synonyms between concepts of different ontologies. For
instance heterogeneity referreds to the variation in notation different e.g. different date
formats.
Merging, aligning and integration is an ontology reuse process to create a new ontology. The
task of each process is as follows (Choi et al., 2006; Ding et al., 2002):
Merging is the task of generating a single ontology by merging two or more different
ontologies of the same domain.
Alignment is a process of creating links between two ontologies when the sources are
consistent but kept separate. This addresses the problem of mapping between ontologies.
Integration generates a single ontology by combining two ore more different ontologies in
different subjects.
Data which covers different domains can not often be described by only one ontology.
Therefore it is necessary to map different ontologies. There are different strategies for
mapping various ontologies:
Ontology mapping between a global ontology and local ontologies (Beneventano et al., 2003):
Denes mapping between concepts in local ontologies to global ontology.
Mapping between local ontologies: These strategies dene mapping between local ontologies.
15
Pellet is a OWL 2 Reasoner for Java (http://clarkparsia.com/pellet).
8.5 Query RDF data
SPARQL overcomes the old problem of different, non standard query languages. Now it is
possible to query RDF data using a standard query language (Quilitz & Leser, 2008). But it is
important that content providers integrate SPARQL-endpoints to make their data available.
Such endpoints provide a machine-friendly interface towards the knowledge base and enables
queries using the SPARQL language. One challenge is to query more than just one endpoint
at the same time with only one query. There are several approaches which can be divided into
two groups (Haase et al., 2010; Kei-Hoi et al., 2009):
Warehousing: This approach stores all RDF data fromthe different resources in one central
database. This database is typically a triple store which is designed to efciently store and
handle RDF data.
Federated query: A query engine decomposes a single query into sub-queries. Each
of these queries can be answered by an individual endpoint. After that, all results are
combined again into one and represented to the user.
Two examples of Java frameworks are Sesame
16
witch supports the warehouse approach and
the ARQ
17
extension of the Jena Ontology API
18
which provides the federated query approach.
8.6 Visualization
The semantic integration of different resources results in increasing the amount of
semantically linked data. Semantic Web technologies use RDF, dening links between data.
Therefore the challenge is to create an interface to visualize and navigate a massive RDF graph
without information overload. The visualization should help the user to easily explore and
quickly nd relevant information (Le Grand & Soto, 2002) in the structure.
8.7 Availability
There are two issues: The availability of ontologies and content. A key to integrating data
using Semantic Web technologies is the availability of ontologies. Many ontologies are freely
available but concerns arise if an ontology is commercial or only partially released. For
example a license is necessary to access UMLS
19
. On the other side it is important to access
content which is annotated to ontologies. But this may cause problems if this content is not
available due to technical problems, deleted static web sites and legal restrictions, etc.
8.8 Different ontology formats
The Semantic Web denes ontologies in the OWL format. But other ontologies exists with
different formats (for example the UMLS Rich Release Format (RRF) or the OBO format).
Therefore, mapping must be dened to convert these different formats to OWL.
8.9 Multilingualism
A challenge is also multilingualism when using Semantic Web technologies (Brner, 2006).
It plays a role in ontology development, annotation of data and representing multilingual
informations in user interfaces (Benjamins et al., 2002). For example, a scenario that leads to a
problem because of multilingualism:
User A annotates a document in French to Term A of an ontology designed in English. User B
16
http://www.openrdf.org/
17
http://jena.sourceforge.net/ARQ/
18
http://jena.sourceforge.net
19
This license is freely available for research purposes
searches for Term A in English and nds a document related to what he is interested in, but it
is written in French.
9. Discussion
The idea behind the Semantic Web is to transform the Web into a global knowledge
base (Kei-Hoi et al., 2009). The key to make this possible is data integration. Therefore
Semantic Web technologies offer a more or less standardized hierarchical framework for data
integration and enable a decentralized semantic integration of different heterogeneous data
sources. For this integration, it is not necessary to change the structure of the data to assemble
knowledge from structured and unstructured sources. This technology extends the source
by adding machine readable semantic metadata using the Resource Description Framework
(RDF). This metadata contains sets of relations between data and concepts. This will
enable people to clearly and commonly dene the concepts and logic within any document
(Neumann et al., 2004). Furthermore, Semantic Web technologies support an automatic
traverse of the connected resources. This queries the integrated sources or even infers new
knowledge using the standard query language SPARQL. The prerequisite for meaningful
semantic data integration is the presence of ontologies. They enable a unique identication
of entities in heterogeneous information systems and provide semantic data integration on
different granular levels. Semantic Web technologies provide standard languages including
the RDF Schema (RDFS), and the Web Ontology Language (OWL) for creating ontologies. The
quality of the data integration is tightly correlated with the quality of the used ontologies. But
in recent years, many high quality open access biomedical ontologies have been created, such
as the Gene Ontology, the Open Biological and Biomedical Ontologies.
In summary, Semantic Web technologies are a promising tool for data integration but there
are still some challenges to be overcome such as uniform naming, extraction of the semantic
information out of existing knowledge, ontology development, ontology maintenance or
query RDF data (see section 8).
10. Additionally
A public available example software, termed OBOBrowsA, can be downloaded following
the link http://www.umit.at/page.cfm?vpath=departments/technik/iebe/
tools/obobrowsa&switchLocale=en_US. It is able to load and display OBO les
20
in
tree or graph representation. The software further allows the user to interactively browse
through the ontology, search for ontology classes and annotate textual data. The manual and
application examples are included in the help function.
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Part 3
Data Mining and Applications

0
Vector Space Information Retrieval Techniques
for Bioinformatics Data Mining
Eric Sakk and Iyanuoluwa E. Odebode
Department of Computer Science, Morgan State University, Baltimore, MD
USA
1. Introduction
Information retrieval (IR) can be dened as the set of processes involved in querying a
collection of objects in order to extract relevant data and information Dominich (2010);
Grossman & Frieder (2004). Within this paradigm, various models ranging fromdeterministic
to probabilistic have been applied. The goal of this chapter is to invoke a mathematical
structure on bioinformatics database objects that facilitates the use of vector space techniques
typically encountered in text mining and information retrieval systems Berry & Browne
(2005); Langville & Meyer (2006).
Several choices and approaches exist for encoding bioinformatics data such that database
objects are transformed and embedded in a linear vector space Baldi & Brunak (1998). Hence,
part of the key to developing such an approach lies in invoking an algebraic structure that
accurately reects relevant features within a given database. Some attention must therefore
be devoted to the numerical encoding of bioinformatics objects such that relevant biological
and chemical characteristics are preserved. Furthermore, the structure must also prove useful
for operations typical of data mining such as clustering, knowledge discovery and pattern
classication. Under these circumstances, the vector space approach affords us the latitude
to explore techniques analogous to those applied in text information retrieval Elden (2004);
Feldman & Sanger (2007); Grossman & Frieder (2004).
While the methods presented in this chapter are quite general and readily applicable to
various categories of bioinformatics data such as text, sequence, or structural objects, we
focus this work on amino acid sequence data. Specically, we apply the BLOCKS protein
sequence database Henikoff et al. (2000); Pietrokovski et al. (1996) as the template for testing
the applied techniques. It is demonstrated that the vector space approach is consistent with
pattern search and classication methodologies commonly applied within the bioinformatics
literature Baldi & Brunak (1998); Durbin et al. (2004); Wang et al. (2005). In addition, various
subspace decomposition approaches are presented and applied to the pattern search and
pattern classication problems.
To summarize, the main contribution of this work is directed towards bioinformatics data
mining. We demonstrate that information measures derived from the vector space approach
are consistent with and, in many cases, reduce to those typically applied in the bioinformatics
literature. In addition, we apply the BLOCKS database in order to demonstrate database
search and information retrieval techniques such as
Pattern Classication
4
Compositional Inferences from the Vector Space Models
Clustering
Knowledge Discovery
The chapter is outlined in Figure 1 as follows. Section 2 provides basic background
regarding information retrieval and bioinformatics techniques applied in this work. Given
this foundation, Section 3 presents various approaches to encoding bioinformatics sequence
data. Section 4 then introduces the subspace decomposition methodology for the vector space
approach. Finally, Section 5 develops the approach in the context of various applications listed
in Figure 1.
Fig. 1. Flowchart for the chapter
2. Overview and notation
Part of the goal of this chapter is to phrase the bioinformatics database mining problem in
terms of vector space IR (information retrieval) techniques; hence, this section is devoted
toward reviewing terms and concepts relevant to this work. In addition, denitions,
mathematical notation and conventions for elements such as vectors and matrices are
introduced.
2.1 Vector space approach to information retrieval
Information retrieval can be thought of as a collection of techniques designed to search
through a set of objects (e.g. contained within a database, on the internet, etc) in order to
extract information that is relevant to the query. Such techniques are applicable, for example,
to the design of search engines, as well as performing data mining, text mining, and text
categorization Berry & Browne (2005); Elden (2004); Feldman & Sanger (2007); Hand et al.
(2001); Langville & Meyer (2006); Weiss et al. (2005). One specic category of this eld that has
proven useful for the design of search engines and constructing vector space models for text
retrieval is known as Latent Semantic Indexing (LSI) Berry et al. (1999; 1995); Deerwester et al.
(1990); Salton & Buckley (1990). Using the LSI approach, textual data is transformed (or
encoded) into numeric vectors. Matrix analysis techniques Golub & Van Loan (1989) are then
applied in order to quantify semantic relationships within the textual data.
for Bioinformatics Data Mining 3
Document 1 Document 2 Document 3 Document 4
Term 1 1 0 1 0
Term 2 0 1 1 0
Term 3 1 1 0 1
Term 4 1 1 0 1
Term 5 1 0 1 0
Table 1. Example of a 5 4 term-document matrix.
Consider categorizing a set of m documents based upon the presence or absence of a list
of n selected terms. Under these circumstances, an n m term-document matrix can be
constructed where each entry in the matrix might reect the weighted frequency of occurrence
of each term of interest. Table 1 provides an example; in this case, a matrix column vector
denes the frequency of occurrence of each term in a given document. Such a construction
immediately facilitates the application of matrix analysis for the sake of quantifying the
degree of similarity between a query vector and the document vectors contained within the
term-document matrix.
Given an n m term-document matrix A, consider an n 1 vector q constructed froma query
document whose components reect the presence or absence of entries in the same list of
n terms used to construct the matrix A. The question then naturally arises how one might
quantify the similarity between the query vector q and the term-document matrix A. Dening
such a similarity measure would immediately lead to a scoring scheme that can be used to
order results from most relevant to least relevant (ie induce a relevance score).
Given the vector space approach, a natural measure of similarity arises from the inner
product. Assuming an
2
-norm, if both q and the columns of A have been normalized to
unit magnitude, then the inner product between q and the j
th
column vector of A becomes
q
T
a
j
= ||q|| ||a
j
|| cos
j
= cos
j
(1)
(where the T superscript denotes the transpose). Since all components of q and A are
non-negative, all inner products will evaluate to a value such that 0 cos
j
1. Similar
queries approach a value of one indicating a small angle between the query and column
vector, dissimilar queries approach a value of zero indicating orthogonality. This specic
measure is called the cosine similarity and is abbreviated as
cos = q
T
A (2)
where cos represents a row vector whose components quantify the relevance between the
query and each column vector of A.
Given the vector space approach, LSI (latent semantic indexing) goes a step further in
order to infer semantic dependencies that are not immediately obvious from the raw data
contained in the term-document matrix. In terms of linear algebra, the LSI methodology
translates into characterizing the column space of A based upon some preferred matrix
decomposition. A tool commonly applied in this arena is the Singular Value Decomposition
(SVD) Golub & Van Loan (1989) where the term-document matrix is factored as follows:
A = UV
T
(3)
87 Vector Space Information Retrieval Techniques for Bioinformatics Data Mining
where U is an n n orthogonal matrix (i.e. U
1
= U
T
), V is an mm orthogonal matrix (i.e.
V
1
= V
T
). Furthermore, is an n m diagonal matrix of singular values such that
1

2

r
> 0 (4)
where r = rank(A) and
i

ii
. It turns out that the rst r columns of U dene an
orthonormal basis for the column space of the matrix A. This basis denes the underlying
character of the document vectors and can be used to infer linear dependencies between them.
Furthermore, it is possible to expand the matrix A in terms of the SVD:
A =
r
j=1
j
u
j
v
T
j
(5)
where u
j
and v
j
represent the j
th
columns of U and V. This expansion weights each product
u
j
v
T
j
by the associated singular value
j
. Hence, if there is a substantial decreasing trend in the
singular values such that
j
/
1
<< 1 for all j > L, one is then led to truncate the above series
in order to focus on the rst L terms that are responsible for a non-negligible contribution to
the expansion. This truncation is called the low rank approximation to A:
A
L
j=1
j
u
j
v
T
j
(6)
The low rank approximation describes, among other aspects, the degree to which each basis
vector in U contributes to the matrix A. Furthermore, the subspace dened by the rst L
columns of U is useful for inferring linear dependencies in the original document space.
2.2 Bioinformatics
Given this abbreviated overview of vector space approaches to information retrieval, we now
put it in the context of bioinformatics research. In particular, the SVD has been applied
in many contexts as it can be thought of as a deterministic version of principal component
analysis Wall et al. (2003). One specic area of honorable mention is pioneering work dealing
with the analysis of microarray data Alter et al. (2000a;b); Kuruvilla et al. (2004).
With regard to information retrieval and LSI in bioinformatics Done (2009); Khatri et al.
(2005); Klie et al. (2008), research in this area devoted to phylogenetics and multiple sequence
alignment Couto et al. (2007); Stuart & Berry (2004); Stuart, Moffett & Baker (2002) has been
reported. Much of this work can be traced back to initial foundations where the encoding
of protein sequences has been performed using the frequency of occurrence of amino acid
k-grams Stuart, Moffett & Leader (2002). Using the k-gram approach, column vectors in the
data matrix (i.e. what was previously referred to as the term-document matrix) are encoded
amino acid sequences and their components are the frequency of occurrence of each possible
k-gram within each sequence. For example, if amino acids are taken k = 3 at a time, then
there exist n = 20
k
= 8000 possible 3-grams. Assuming there are m amino acid sequences, the
associated data matrix will be n m = 8000 m. For each amino acid sequence, a sliding,
overlapping window of length k is used to count the frequency of occurrence of each k-gram
and entered into the data matrix A.
The goal of this chapter is to build upon the IR and bioinformatics foundation in order
to introduce novel perspectives on operations and computations commonly encountered in
bioinformatics such as the consensus sequence, position specic scoring matrices (PSSM),
database searches, pattern classication, clustering and multiple alignments. In doing so, it
is our intent that the readers view of these tools will be expanded toward novel applications
beyond those presented here.
3. Sequence encoding
Many choices exist for the encoding of and weighting of entries within the term-document
matrix; in addition, there exist a wide range of possibilities for matrix decompositions as
well as the construction of similarity and scoring measures Elden (2004); Feldman & Sanger
(2007); Hand et al. (2001); Weiss et al. (2005). The goal of this chapter is not to expand on the
set of choices for the sake of text retrieval and generic data mining; instead, we must focus
on techniques and approaches that are relevant to bioinformatics. Specically, our attention
in this section is devoted toward developing and presenting novel encoding schemes that
preserve relevant biological and chemical properties of genomic data.
An assortment of methods have been proposed and studied for converting a protein from
its amino acid sequence space into a numerical vector Bacardit et al. (2009); Baldi & Brunak
(1998); Bordo & Argos (1991); Stuart, Moffett & Leader (2002). Scalar techniques generally
assign a real number that relates an amino acid to some physically measurable property (e.g. -
volume, charge, hydrophobicity) Andorf et al. (2002); Eisenberg et al. (1984); Kyte & Doolittle
(1982); Wimley & White (1996). On the other hand, orthogonal or standard vector encoding
techniques Baldi & Brunak (1998) embed each amino acid into a k dimensional vector space
where k is the number of symbols. For example, if k = 20 (as it would be for the complete
amino acid alphabet), the j
th
amino acid where 1 j 20 is represented by a 20 dimensional
vector that is assigned a one at the j
th
position and zero in every other position. In general,
standard encoding transforms a sequence of length L into an n = Lk dimensional vector. As
an example consider the DNA alphabet A = {A, G, C, T}. In this case k = 4 and standard
encoding transforms the alphabet symbols as
A =
1
0
0
0
. G =
0
1
0
0
. C =
0
0
1
0
. T =
0
0
0
1
. (7)
Therefore, for an example sequence s = AT with L = 2, this encoding method yields the
following vector of dimension n = Lk = 8:
x
T
=
1 0 0 0 0 0 0 1
.
Observe that, for typical values of L, assuming a data set of m sequences, standard encoding
leads to an n m data matrix that is sparse.
In bioinformatics, given the limitations on biological measurement, the number of
experimental observations tends to be limited and values of m are often small with respect
to n. Under these conditions, it is often the case that vector encoding methodologies lead
to sparse data matrices (as is the case for text retrieval applications) in high dimensional
vector spaces. Observe, for example, that the k-gram method reviewed in Section 2.2 ts
this description.
We can expand upon the standard encoding approach by categorizing the standard amino
acid alphabet into families that take into account physical and chemical characteristics derived
from the literature Andorf et al. (2002); Baldi & Brunak (1998). In addition, entries within
the data matrix can be weighted based upon their hydrophobicity Eisenberg et al. (1984);
Kyte & Doolittle (1982). Table 2 introduces alphabet symbols used to group amino acids
according to hydrophobicity, charge and volume. Tables 3-5 show examples of various
encoding schemes that we apply for this analysis.
Hydrophobicity R=hydrophobic, H=hydrophilic
Charge P=positive, N=negative, U=uncharged
Volume S=smal, M=medium, ML=medium-large, L=medium
Table 2. Encoding symbols applied in Tables 3-5
R 1 A, I, L, M, F, P, W, V, D, E
H 3 R, H, K, N, C, Q, G, S, T, Y
Table 3. Hydrophobic/Hydrophilic Encoding
RU 1 A, I, L, M, F, P, W, V
HN 2 D, E
HP 3 R, H, K
HU 4 N, C, Q, G, S, T, Y
Table 4. Charged Hydrophobic/Hydrophilic Encoding
RUS 1 A
RUM 2 F
RUML 3 I, L, M, V
RUL 4 F, W
HPML 5 R, H, K
HNM 6 D
HNML 7 E
HUS 8 G, S
HUM 9 N, C, V
HUML 10 Q
HUL 11 Y
Table 5. Volume/Charged Hydrophobic/Hydrophilic Encoding
4. Subspace decompositions for pattern classication
LSI techniques necessarily require the application of matrix decompositions such as the SVD
to infer column vector dependencies in the data matrix. Decompositions of this kind can
lead to the construction of subspaces that can mathematically categorize subsets of sequences
into families. Furthermore, since these families dene specic classes of data, they can be
used as training data in order to perform database searches and pattern classication. The
application of linear subspaces for the sake of pattern classication Oja (1983) consists of
applying orthogonal projection operators based upon the training classes (an orthogonal
projection operator P obeys P = P
T
and P
2
= P).
4.1 Orthogonal projections
To begin, let us assume there are training sequences of known classication that can be
categorized into M distinct classes and that the i
th
class contains m
i
encoded vectors of
dimension n. For each class, an n m
i
matrix A
i
can be constructed (assuming the training
vectors are column vectors). To characterize the linear subspace generated by each class, we
can apply the singular value decomposition (SVD) Golub & Van Loan (1989). In addition to
providing us with an orthonormal basis for each class, we can also glean some information
about the inuence of the singular values and singular vectors from the rank approximants.
Class data matrices are therefore decomposed as
A
i
= U
i
i
V
T
i
(8)
where U
i
is n n orthogonal matrix,
i
is n m
i
whose diagonal contains the singular values
and V
i
is an m
i
m
i
orthogonal matrix. Assume the rank of each data matrix A
i
is r
i
and let
Q
i
denote the n r
i
matrix formed from rst r
i
columns of U
i
. Given the properties of the
SVD, the columns of Q
i
dene an orthonormal basis for the column space of A
i
. Hence, an
orthogonal projection operator for the i
th
class is established by computing
P
i
= Q
i
Q
T
i
. (9)
(given that the SVD induces U
T
i
= U
1
i
, it is straightforward to check that P
2
i
= P
i
and
P
T
i
= P
i
).
Consider an n 1 query vector x whose classication is unknown. The class membership of
x can be ascertained by identifying the class yielding the maximum projection norm:
C(x) argmax
i=1, , M
||P
i
x||. (10)
One computational convenience of constructing the orthonormal bases Q
i
is that it is not
necessary to compute the projections when making this decision. Given any Q with
orthonormal columns and orthogonal projection P = QQ
T
such that P
2
= P and P = P
T
,
observe that
||Px||
2
= x
T
P
T
Px = x
T
P
2
x
= x
T
Px = x
T
QQ
T
x
= ||Q
T
x||
2
= ||x
T
Q||
2
.
(11)
Under these circumstances, to decide class membership, Equation (10) reduces to
C(x) argmax
i=1, , M
||x
T
Q
i
||. (12)
Furthermore, the values ||x
T
Q
i
|| immediately yield relevance scores and condence measures
for each class.
4.2 Characterization of the orthogonal complement
It is important to note that the union of all the class subspaces need not be equal to the n
dimensional vector space from which all data vectors are derived. To perform a complete
orthogonal decomposition of the n dimensional vector space in terms of the data, we rst
dene the matrix
A [A
1
A
M
]. (13)
The goal then is to characterize the null space N(A
T
), the subspace which is orthogonal to the
column space of A. Assuming the rank of A is r
A
, computing the SVD
A = U
A
A
V
T
A
(14)
and forming the matrix Q
A
from the the rst r
A
columns of U
A
yields an orthogonal
decomposition of the subspace generated by all class vectors. Hence, a projection operator
for this subspace is constructed as
P
A
= Q
A
Q
T
A
. (15)
In addition, a projection for the orthogonal complement N(Q
T
A
) of A is then easily formed via
P
A
= I
n
P
A
(16)
where I
n
is the n n identity matrix. A complete orthogonal decomposition Lay (2005) of a
vector x R
n
can then be determined from
x = P
A
x + P
A
x. (17)
4.3 Information retrieval
Before attempting to decide the class membership of a vector x R
n
based upon Equation
(12), it is sensible to characterize the portion of the vector that contributes to the class subspace
dened by Q
A
. Given Equation (17), this is most easily done by comparing ||P
A
x|| with
||P
A
x|| as
tan() =
||P
A
x||
||P
A
x||
(18)
where is the angle between x and the subspace dened by Q
A
. Ideally, if the class
subspaces have been completely characterized, tan() should be small. Conversely, larger
values of tan() would indicate that x is a member of a class subspace that has not yet been
dened. Under these circumstances, the orthogonal complement would have to be further
characterized and partitioned in order to dene more classes beyond the known M existing
classes.
It is also possible to phrase the tangent measure as a scalar version of the more familiar cosine
similarity dened above in Equation (2). If ||x|| = 1, the cosine similarity measure takes on a
convenient form
cos() = ||x
T
Q
A
||. (19)
To see why, consider the inner product
x
T
(P
A
x) = x P
A
x = ||x|| ||P
A
x|| cos(). (20)
If ||x|| = 1, then
cos() =
x
T
P
A
x
||P
A
x||
. (21)
However, since P
A
is an orthogonal projection
||P
A
x|| =
x
T
P
T
A
P
A
x =
x
T
P
A
x (22)
and Equation (21) can therefore be rewritten as
cos() =
x
T
P
A
x. (23)
On the other hand, by applying Equation (11) to Equation (22), it follows that
||x
T
Q
A
|| =
x
T
P
A
x (24)
as well; hence, the equality of Equations (23) and (24) establishes Equation (19).
Equation (19) should also be clear from the geometric fact that
cos() =
||P
A
x||
||x||
. (25)
Assuming ||x|| = 1, Equation (19) then easily follows by applying Equation (11) to Equation
(25). Equations (23) and (24) are presented in order to offer additional insight by relating the
inner product to the projection operator.
Of central focus in the next section will be to apply the above projection framework to
information retrieval in bioinformatics. Since the classication problemwill be of signicance,
we note that, given the identity in Equation (19), Equation (12) can be rephrased in termof the
cosine similarity measure
C(x) argmax
i=1, , M
cos(
i
) (26)
where
cos(
i
) ||x
T
Q
i
||. (27)
In addition, this measure of class membership becomes more reliable if the contribution of x
to the orthogonal complement of the data set is small. For instance, when is small, cos()
in Equation (19) approaches unity. Therefore, cos() can be applied as a measure of data
set reliability while cos(
i
) can be used to produce relevance scores for i = 1, , M. These
conclusions are summarized in Table 6.
Similarity Measure Purpose Reference
cos() Data Set Reliability Equation (19)
cos(
i
) Relevance Score Equations (26) - (27)
Table 6. Reliability and relevance measures to be applied in Section 5
.
5. Applications
In bioinformatics, families with similar biological function are often formed from sets of
protein or nucleic acid sequences. For example, databases such as Pfam Finn et al. (2010),
PROSITE Sigrist et al. (2010) and BLOCKS Pietrokovski et al. (1996) categorize sequence
domains of similar function into distinct classes. Given the encodings discussed in Section 3,
we seek to demonstrate how Equations (19) and (26) can applied in order to performsequence
modeling, pattern classication and database search computations typically encountered in
bioinformatics Baxevanis & Ouellette (2005); Durbin et al. (2004); Mount (2004).
5.1 Consensus sequence
A set of m sequences of length L having some related function (e.g. DNA promoter sites
for a common sigma factor) is often represented in the form of an m L matrix where each
column refers to a common position in each sequence. A consensus sequence s
C
of length
L is constructed by extracting the symbol having the highest frequency in each column.
This approach to sequence model construction, while quite rudimentary, is often useful for
visualizing obvious qualitative relationships amongst sequence elements.
Using the vector space approach, it is possible to recover the consensus sequence. Assuming
each sequence symbol is encoded into a k dimensional vector, each sequence will be encoded
into a vector of length n = Lk (see Section 3). Hence the original m L matrix of sequences
will be transformed into an n mdata matrix of the formdescribed in Section 2.1. In this case,
each column vector in the data matrix represents an encoded amino acid sequence.
To recover the consensus, it is useful to introduce notation for describing an empirically
derived average vector
A
from an n m data matrix A as follows:
A
(
1
m
)Ae (28)
where e and m1 vector of ones. Then,
A
is an n 1 column vector made up of L contiguous
subvectors of dimension k where the value of k depends upon the encoding method applied.
Let
i
for i = 1, , L represent each subvector in
A
; then, the i
th
symbol in the consensus
sequence s
C
(i) can be inferred by associating the component of
i
yielding the highest average
with the originally encoded symbol. To be precise, let the alphabet of k sequence symbols (e.g.
DNA, amino acids, structural, text, etc) be dened as
A {a
1
, a
2
, , a
k
} (29)
and let the j
th
component of
i
be written as
ij
for j = 1, , k. The subscript index of the
component with the maximum average in
i
can therefore be extracted as
J = argmax
j=1, ,k
ij
(30)
and the associated alphabet symbol is entered into the i
th
position of the consensus sequence
as
s
C
(i) = a
J
(31)
where a
J
A. The algorithm for recovering the consensus sequence can be summarized as
follows:
1. Given the n m encoded data matrix A, compute
A
.
2. For each
i
where i = 1, , L, apply Equation (30).
3. Given the alphabet A, apply Equation (31) in order to construct the consensus sequence
s
C
.
5.2 Position specic scoring matrix
The consensus sequence, while qualitatively useful, is an incomplete sequence model in
that it does not consider cases where two or more symbols in a given position are close to
equiprobable. Under these circumstances, one is forced to arbitrarily choose one symbol for
the consensus at the expense of loosing information about the other symbols. In contrast,
the position specic scoring matrix (PSSM) is a sequence model that considers the frequency
of occurrence of all symbols in each position. Furthermore, the PSSM can be used to score
and rank sequences of unknown function in order to quantify their similarity to the sequence
model.
Given an m L matrix of of m related sequences of length L and an alphabet of k symbols, a
k L prole matrix of empirical probabilities is rst constructed by computing the symbol
frequency for each position. The prole matrix can be thought of as the preimage of the PSSM.
While it can provide important statistical details regarding the sequence model, it does not
have the capability to score sequences in an additive fashion position by position. To do
this requires converting the prole into a k L PSSM of additive information scores. Given
a sequence s of length L, the PSSM can then be used to compute a score for s in order to
determine its relationship to the sequence model.
Recovering the PSSM fromthe vector space approach is straightforward. Given an n m data
matrix of encoded sequences, the i
th
subvector
i
in the average vector
A
computed from
Equation (28) is equivalent to the i
th
column in the k L prole matrix. Simply reshaping the
kL 1 vector
A
into a k L matrix recovers the prole. However, since the goal is to score
sequences of unknown function, we are more interested in showing how
A
can be applied to
recover a PSSMscore. Assume that the components of
A
have been transformed by applying
the same information measure I
PSSM
used to convert the prole to the PSSM. Assuming an
encoding alphabet with k symbols, a query sequence s of length L can be encoded to form a
kL 1 vector x. The PSSM score S
PSSM
of x can then be recovered via the inner product:
S
PSSM
= x
T
I
PSSM
(
A
) (32)
where I
PSSM
(
A
) represents the conversion of a probability vector into an vector of additive
information scores.
The similarity of Equation (32) with Equation (2) is worth noting. Assume several families of
sequences of equal length L are encoded into separate data matrices A
i
where i = 1, , M
and M is the number of families. It should be clear that the relevance score for the query
vector x can be produced using the cosine similarity according to
S
PSSM
= x
T
I
PSSM
(33)
where
I
PSSM
=
I
PSSM
(
A
1
) I
PSSM
(
A
2
) I
PSSM
(
A
M
)
(34)
is the n M information matrix that describes the sequence families.
It is of important theoretical interest that the vector space approach recovers both the PSSM
and its information capacity to score sequences. However, it is more useful to observe
that invoking an algebraic structure on a set of sequences induces a spectrum of novel
possibilities. For instance, the SVD can be applied to the data matrix and a scoring scheme
can be derived from the computed orthogonal basis. In addition, as mentioned at the end of
Section 3, it is possible to weight both the data matrix and the encoded sequence according
to more biologically signicant measures such as hydrophobicity. Finally, and probably
most importantly, the vector space formulation allows for powerful optimization techniques
Golub & Van Loan (1989); Luenberger (1969) to be applied in order to maximize the scoring
capacity of the sequence model.
5.3 Clustering
Our goal in this section is to investigate how clustering encoded sets of vectors will partition
an existing set of data. While there are several approaches to performing data clustering
Theodoridis & Koutroumbas (2003), we choose to invoke techniques that characterize the
mean behavior of a data cluster. Specically, we analyze one supervisedmethod (Section 5.3.2)
and one unsupervised method (Section 5.3.3). As we shall see, these approaches will enable
us to construct fuzzy regular expressions capable of algebraically describing the behavior
of a given data set. It will become clear that this approach will offer additional insight to
sequence clustering techniques typically encountered in the literature Henikoff & Henikoff
(1991); Smith et al. (1990). As the BLOCKS database Henikoff et al. (2000); Pietrokovski et al.
(1996) has been constructed from sequence clusters using ungapped multiple alignment, we
choose to apply this database as the template in order to compare it against the vector space
model.
5.3.1 The BLOCKS database
The BLOCKS database consists of approximately 3000 protein families (or blocks). Each
family has a varying number of sequences that have been derived fromungapped alignments.
Therefore, while sequence lengths between two different families may differ, sequences
contained within each family, by the denition of a block, must all have the same length.
Furthermore, the number of sequences in each family can vary and there is can be a
considerable degree of redundancy within some families; hence, it is sensible to analyze how
the data is distributed with respect to each BLOCKS family.
The histogram in Figure 2 illustrates the number of BLOCKS families as function of sequence
length. For example, there are 90 families containing sequences of length L = 40. From this
gure, we can conclude that it is generally possible to nd at least 40 families containing
nominal sequence lengths. It is also important to characterize how the number of sequences
contained within each family is distributed throughout the database. The histogram in Figure
3 illustrates the number of BLOCKS families as function of the number of sequences contained
within each family. From this gure, we observe that many families contain somewhere
between 9 and 20 representative sequences. Finally, for the sake of clarity, we restrict our
attention to sequences having the same lengths. The extension of these results to variable
length sequences is the subject of current research based upon existing methodologies cited in
the literature Couto et al. (2007); T. Rodrigues (2004). The histogram in Figure 4 illustrates the
number of BLOCKS families as function of the number of sequences contained within in each
family; however, observe that this representative sample has been restricted to those families
containing sequences of equal length (in this case L = 30). The behavior in this graph is typical
in that most families contain on the order of 10-12 sequences of equal length. For the purposes
of illustration and without loss of generality, we choose to demonstrate the techniques in the
upcoming sections using families containing sequences of equal length.
5.3.2 Centroid approach
In this section, we cluster sequences whose BLOCKS classication is known a priori in order
to algebraically characterize each family. To do this, each family in the analysis is encoded
separately and Equation (28) is applied to each family data matrix in order to derive a family
centroid. Since the families are already partitioned, this approach is a supervised clustering
technique that will enable us to derive symbol contributions from the centroid vectors.
0 10 20 30 40 50 60
0
10
20
30
40
50
60
70
80
90
100
Sequence length, L
N
u
m
b
e
r

o
f

B
L
O
C
K
S

f
a
m
i
l
i
e
s
Fig. 2. Histogram of the number of BLOCKS families as function of sequence length.
0 10 20 30 40 50
0
100
200
300
400
500
600
700
Number of sequences
N
u
m
b
e
r

o
f

B
L
O
C
K
S

f
a
m
i
l
i
e
s
Fig. 3. Histogram of the number of BLOCKS families as function of the number of sequences
contained in each family.
0 5 10 15 20 25
0
2
4
6
8
10
12
14
16
18
20
Number of sequences
N
u
m
b
e
r

o
f

B
L
O
C
K
S

f
a
m
i
l
i
e
s
Restricted to Families with Sequence Length L=30
Fig. 4. Histogram of the number of BLOCKS families as function of the number of sequences
contained in each family (restricted to families with sequences of length L=30)
For this numerical experiment, we apply Table 5 as the encoding scheme and choose the
BLOCKS family sequence length to be L = 30. Under these conditions, sequences will be
encoded into column vectors of dimension n = (30)(11) = 330. In addition, all encoded data
vectors are normalized to have unit magnitude.
There are 73 families in the BLOCKS database that have block length L = 30. Furthermore,
there are a total of 910 sequences distributed amongst the 73 families. As mentioned above,
there is a small degree of sequence redundancy within some BLOCKS families. After
removing redundant sequences, a total of J = 755 sequences of length L = 30 are distributed
amongst I = 73 families. Given the encoding method, the dimensions of the non-redundant
data matrix A will be 330 755.
Figure 5 shows the results of computing the distance between all centroids. From this
histogram, we observe that database families are fairly well-separated since the minimum
distance between any two centroids is greater than 0.6.
In order to analyze the performance of the encoding method, we apply the inner product.
Specically, each data vector v
j
is classied by choosing the family associated with the
centroid yielding the largest inner product:
C(v
j
) argmax
i=1, , I
v
T
j
M. (35)
where j = 1, , J and
M=
A
1

A
2

A
J
(36)
For standard encoding (i.e. k = 20, n = 600), all 755 data vectors were classied correctly
using Equation (35). On the other hand, when applying the encoding method in Table 5, there
0.6 0.8 1 1.2 1.4 1.6 1.8 2
0
10
20
30
40
50
60
70
80
90
Between Centroid Distance (L=30, 73 Families)
F
r
e
q
u
e
n
c
y
Fig. 5. Histogram of between centroid distance.
was one misclassication. Figure 6 illustrates that data vector number 431 (which as member
of family 30, HlyD family secretion proteins) was misclassied into family 54 (Osteopontin
proteins). So, while the vector dimension is reducedfrom600 to 330 (because k is reducedfrom
20 to 11), a minor cost in classication accuracy is incurred. At the same time, we observe a
substantial reduction in dimensionality.
We note one nal application of the centroid approach for deriving fuzzy regular expressions
extracted from the vector components of the centroid vectors. Consider the sum normalized
i
th
family centroid
N
A
i

n
j=1
(
A
i
)
j

A
i
. (37)
For each subvector associated with each sequence position in N
A
i
, it is then possible to
write an expression describing the percentage contribution of each symbol to analytically
characterize the i
th
sequence family.
5.3.3 K-means approach
In contrast to the supervised approach, we now wish to take all sequences of length L in the
database and investigate how they are clustered when the unsupervised K-means algorithm
is applied. When this algorithmis applied to small numbers of families (e.g. < 10), our results
indicate that this algorithm will accurately determine the sequence families for the encoding
method presented. However, as the number of data vectors grow, the high-dimensionality
of the encoding method tends to obscure distances and, hence, can obscure the clusters. We
briey address this issue in the conclusions section of this chapter.
0 100 200 300 400 500 600 700 800
0
10
20
30
40
50
60
70
80
Data Vector Index (L=30)
C
l
a
s
s
i
f
i
c
a
t
i
o
n

b
y

F
a
m
i
l
y

I
n
d
e
x
Fig. 6. Family classication of each data vector.
5.4 Database search and pattern classication
We now come to what is arguably one of the most important applications in this chapter.
In this section, we will apply the reliability and relevance measures summarized in Table 6
to perform BLOCKS database searches and pattern classication Bishop (2006); Hand et al.
(2001).
5.4.1 Characterization of BLOCKS orthogonal complement
When constructing a database, it is critical to understand and analytically characterize the
spectrum of objects not contained within the database. This task is easily achieved by
considering the orthogonal complement. As rst step, we consider families with sequence
lengths L = 15 (70 families) and L = 30 (73 families). Furthermore, we compare encodings
from Table 3 and Table 5 with standard encoding. Specically, for each encoding method,
an n m non-redundant data matrix A consisting of all data vectors of from all families with
sequence length L is constructed. The SVDis then applied to construct an orthogonal basis Q
A
for the column space of A. The rank r of A(r=D[Q
A
]) and the dimension of the null space of A
are then compared (D[N(Q
T
A
)]). Using this approach, it is then possible to assess the quantity
n D[Q
A
] to determine the size of the subspace left uncharacterized by the database. Table
7 summarizes the results. From this table, it is clear that, after redundant encoded vectors
are removed, the BLOCKS database thoroughly spans the pattern space. Furthermore, the
histogram in Figure 5 further indicates that, while the sequence subspace is well represented,
there is also a good degree of separation between the family classes.
5.4.2 Pattern classication
Another important database characterization is to examine how the projection method
classies data vectors after the class subspace bases have been constructed using the SVD.
renewcommand11.2
Sequence Length Encoding Method n m D[Q
A
] D[N(Q
T
A
)]
L = 15 Standard 300 949 286 14
L = 15 Table 5 165 949 165 0
L = 15 Table 3 30 936 30 0
L = 30 Standard 600 785 576 13
L = 30 Table 5 330 785 330 0
L = 30 Table 3 60 774 60 0
Table 7. Characterization of BLOCKS orthogonal complement for various sequence lengths
and encodings
In a manner similar to Figure 6, we classify all encoded data vectors in order to determine
their family membership by applying Equation (26). Figures 7 - 8 show results where the
L = 15 and L = 30 cases have been tested. For the L = 15 case, as the vector space
dimension decreases more classication errors arise since a reduced encoding will result in
more non-unique vectors. The L = 30 case leads to longer vectors, hence, it is more robust to
reduced encodings.
0 200 400 600 800 1000
0
50
Vector Index (Standard Encoding, L=15)
F
a
m
i
l
y

I
n
d
e
x
0 200 400 600 800 1000
0
50
Vector Index (Volume/Charge/Hydro Encoding, L=15)
F
a
m
i
l
y

I
n
d
e
x
0 200 400 600 800 1000
0
50
Vector Index (Hydro Encoding, L=15)
F
a
m
i
l
y

I
n
d
e
x
0 100 200 300 400 500 600 700 800
0
50
Vector Index (Standard Encoding, L=30)
F
a
m
i
l
y

I
n
d
e
x
0 100 200 300 400 500 600 700 800
0
50
Vector Index (Volume/Charge/Hydro Encoding, L=30)
F
a
m
i
l
y

I
n
d
e
x
0 100 200 300 400 500 600 700 800
0
50
Vector Index (Hydro Encoding, L=30)
F
a
m
i
l
y

I
n
d
e
x
5.4.3 BLOCKS database search
In this section, we demonstrate how to perform database searches using the relvance and
reliability equations summarized in Table 6. Database search examples have been reported
using the BLOCKS database Henikoff & Henikoff (1994). In this work, we analyze the effect of
randomly mutating sequences within the BLOCKS database to analyze family recognition as a
function sequence mutation. For the purposes of illustration, we consider a test sequence from
the Enolase protein family (BL00164D) in order to examine relevancy and database reliability.
For this test sequence with L = 15, amino acids are randomly changed where the number of
positions mutated is gradually increased from 0 to 12. Furthermore, encodings from Table 3
are compared with standard encoding.
For this series of tests, the reliability always gives a value of cos() = 1, implying that the
randomization test did not result in a vector outside the subspace dened by the database.
This corroborates conclusions drawn in Section 5.4.1. Figure 9 shows that the classication
remains stable for both encodings until about 5-6 positions out of 15 have been mutated (the
family index for the original test sequence is 10). In addition, the relevance can be summarized
by computing the difference between the maximum value of cos(
i
) and the second largest
value. For the sake of illustration, if the BLOCKS family with index 10 does not yield the
maximum projection, then the relevance difference is assigned a negative value. Figure 10
show the results of this computation. In this test, we observe a consistent decrease in the
relevance difference indicating that secondary occurrences are gaining inuence against the
family class of the test sequence.
0 2 4 6 8 10 12
6
8
10
12
Number of Positions (Standard Encode)
F
a
m
i
l
y

I
n
d
e
x
0 2 4 6 8 10 12
5
10
15
20
Number of Positions (Volume/Charge/Hydro Encode)
F
a
m
i
l
y

I
n
d
e
x
Fig. 9. Family classication as a function of the number of positions randomized.
0 2 4 6 8 10 12
0.5
0
0.5
1
Number of Positions (Standard Encode)
R
e
l
e
v
a
n
c
e

D
i
f
f
e
r
e
n
c
e
0 2 4 6 8 10 12
0.5
0
0.5
1
Number of Positions (Volume/Charge/Hydrod Encode)
R
e
l
e
v
a
n
c
e

D
i
f
f
e
r
e
n
c
e
Fig. 10. Relevance differential as a function of the number of positions randomized.
6. Conclusions
This chapter has elaborated upon the application of information retrieval techniques to
various computational approaches in bioinformatics such as sequence modeling, clustering,
pattern classication and database searching. While extensions to multiple sequence
alignment have been alluded to in the literature Couto et al. (2007); Stuart, Moffett & Baker
(2002), there is a need to include and model gaps in the approaches proposed in this body of
work. Extensions to the vector space methods outlined in this chapter might involve including
a new symbol to represent a gap. Regardless of the symbol set employed, it is clear that the
approach described can lead to sparse elements embedded in high dimensional vector spaces.
While data sets of this kind can be potentially problematic Beyer et al. (1999); Hinneburg et al.
(2000); Houle et al. (2010); Steinbach et al. (2003), subspace dimension reduction techniques
are derivable from LSI approaches such as the SVD.
The IR techniques introduced above are readily applicable in any setting where bioinformatics
data (sequence, structural, symbolic, etc) can be encoded. This work has focused primarily
on amino acid sequence data; however, given existing structural encoding techniques
Bowie et al. (1991); Zhang et al. (2010), future work might be directed toward vector
space approaches to structural data. The methods outlined in this chapter allow for
novel biologically meaningful weighting schemes, algebraic regular expressions, matrix
factorizations for subspace reduction as well as numerical optimization techniques applicable
to high dimensional vector spaces.
7. Acknowledgements
This work was made possible by funding from grant DHS 2008-ST-062-000011, Increasing
the Pipeline of STEM Majors among Minority Serving Institutions. The authors would like
to thank David J. Schneider of the USDA-ARS for many helpful discussions.
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5
Massively Parallelized
DNA Motif Search on FPGA
Yasmeen Farouk
1
, Tarek ElDeeb
2
and Hossam Faheem
1

1
Faculty of Computer and Information Sciences, Ain Shams University,
2
Faculty of Engineering, Cairo University,
Egypt
1. Introduction
Understanding the mechanisms that regulate gene expression is a major challenge in
biology. Motif finding problem is considered an important task in this challenge.
Addressing the complexity nature of the problem together with being very data intensive
has encouraged introducing field programmable gate arrays (FPGAs) to the problem.
FPGAs are very powerful in such computationally intensive tasks.
Many Algorithms are introduced to solve this problem. They can be categorized into
pattern-based and profile-based algorithms [1]. Pattern-based algorithms include
PROJECTION[4], MULTIPROFILER[6], and MITRA[3]. Profile-based algorithms includes
CONSENSUS[7], MEME[2] and Gibbs sampling[5]. Although these algorithms show good
performance, they still can fail to identify all the possible motifs in the sequences. They also
show poor performance when trying to solve the challenge problem presented by Pvzner
and Sze[8]. Some of them fail due to local search, others which are based on statistical
measures fail to separate the motif from the background sequences.
We can also categorize Motif finding algorithms due to the solution they provide. Some
algorithms provide exact solution others provide approximate one. Brute Force algorithm is
an exact algorithm but it suffers from the intractability of its running time. It increases
exponentially with the size of the required motif. This makes the Brute Force unsuitable for
long motifs.
Our enhanced Brute Force algorithm, skip Brute Force, can predict the quality of the
computed motif. The algorithm skips those iterations which will lead to a poor scored motif,
thus leads to a better running time than the original Brute Force. This enhancement
guarantees the same exactness of the Brute Force. But, it still suffers from the intractable
running time for long motifs.
Many approaches can be applied to speed up the running time of any algorithm using
hardware; examples include chip multiprocessors, graphics processing units (GPUs) and
(FPGAs). GPUs are inexpensive, commodity parallel devices and have already been
employed as powerful coprocessors for a large number of applications. However, GPUs
have limited instructions and limited parallelism relative to FPGA's configurability. The
research in [10] employed acceleration using GPU. Another approach uses clusters of
workstations [12]. However, clusters typically have high maintenance and energy costs

Bioinformatics Trends and Methodologies 108
when compared to single node solutions. Others use special hardware [9][11], where a cost
performance ratio would be fairer for comparison [9].
The repetitive nature of the algorithm and the locality of the data encourage the use of
FPGAs. Many operations can be done concurrently to enhance the running time. FPGAs
proved to successfully accelerate sequential algorithms minimum by one or two orders of
magnitude. They also have been widely used to accelerate bioinformatics problems such as
Smith-Waterman and BLAST algorithms. This research offers an enhanced Brute Force
algorithm hardware accelerated using Field Programmable Gate Arrays (FPGAs). Our
research leads to a speed up by 1.5MX and thus boosting the running time without
sacrificing the accuracy.
The rest of this chapter is organized as follows: In Section 2 we describe the motif finding
problem and presents our enhanced Brute Force algorithm; skip Brute Force. Section 3
presents the hardware implementation of our novel approach with a detailed view to its
components. Performance evaluation is presented in section 4. Finally, section 5 concludes
our work and presents future enhancements.
2. Skip brute force algorithm
Brute-force search or exhaustive search, also known as generate and test, is a very general
problem solving technique that consists of systematically enumerating all possible
candidates for the solution and checking whether each candidate satisfies the problem's
statement.
The motif finding problem can be summarized as follows:
Planted (l,d)- Motif Problem: Find the motif consensus M which is a fixed but unknown
nucleotide sequence of length l. Suppose that M occurs once in each of t background
sequences of common length n. Each occurrence of M is mutated by exactly d point
substitutions in positions chosen independently at random. Given the t sequences, recover
the motif occurrences and the consensus M.
Pevzner and Sze[8] presented the challenge problem(15,4) which makes a particular
parameterization to the panted motif problem. The motif we are searching for is of length
l=15, the allowed mutations d=4 and the number of sequences we are searching in is t=20
each of size n=600. The parameters of the challenge problem are typical values for finding
transcription factor binding sites in co-regulated gene promoter regions yeast [4].
The Brute Force algorithm solves the motif finding problem by considering the set of all 4
l

possible l-mers. It computes the total distance of each l-mer in that set to all other l-mers in all t
sequences. The correct motif is the one that have the smallest total distance along all the other
l-mers. The run time of this algorithm is O(4
l
nt). The running time for finding a motif of l=11 is
about 5hrs and it fails to handle longer motifs in reasonable time. To solve the challenge
problem, the running time of the Brute Force algorithm would obviously be too slow.
The idea behind our skip Brute Force algorithm is that it skips all the iterations that will not
lead to a correct solution. The algorithm is forced to skip over the remaining iterations in two
cases. The algorithm generates all possible 4
l
l-mers. It then iterates over all the sequences
examining that generated l-mer with all the windows in each sequence. For each sequence
iteration, the current score is initialized with the allowed mutation and then the score of each
window is computed; i.e. the hamming distance between that window and the current l-mer.
If this distance beats the current score then we would suspect the current window to be an
implanted motif until another window in the same sequence with a higher score beats it.

Massively Parallelized DNA Motif Search on FPGA 109
The planted motif problem guarantees to find the motif in each sequence. Based on this fact
the skip algorithm skips the iterations over the remaining sequences if it reached the end of
the current sequence without finding any window that matches the current l-mer (this l-mer
can not be the motif) and jumps to the next l-mer. Assuming a single solution, the algorithm
also skips the iterations over the remaining l-mers if it reaches the last sequence (t=20)
without skipping any iteration (the solution is found).
A pseudo code of the skip Brute Force algorithm is shown below in Figure 1.

Fig. 1. Pseudo Code of the skip Brute Force Algorithm. If the commented break command is
applied, then algorithm will skip-more.
2.1 Skip-more brute force
In our early implementation of the skip algorithm, we did not consider scores for the motifs
found. We forced to skip the current sequence if a single motif is found that has d mutations
within the allowed range (line 15). Here the algorithm fails to find the best motif as more
windows in the current sequence can reveal occurrences of motifs with lower mutations.
The complexity of this algorithm is O(4
l
nt) at its worst case, just as the Brute Force.
3. Hardware implementation of skip brute force
Our design benefits from the concurrent nature of the FPGAs as a hardware platform;
control, multiplexing, matching and decision making are all occurring on the same clock

edge. We used VHDL to model our design preserving its extendibility for more complex
challenging problems in future. Figure 2 shows the system block diagram.

Fig. 2. Block diagram of the skip Brute Force - running on an FPGA with one matching unit.
All t-sequences are first loaded into an on-chip read-only memory 'ROM' as shown in Figure
3. On the contrary, the set of all 4
l
l-mers are not stored, but locally generated. Gaining from
encoding each nucleotide into 2-bit symbol, the 4
l
Motif Generator is a simple controlled
binary counter. The shifter block is fed by the currently needed sequence and only reveals a
sliding l-sized window of it at a time. The matching block compares the revealed window to
the generated l-mer and outputs the hamming distance as the mutation score. The logical
control unit synchronizes the system to properly implement the skip Brute Force algorithm.
More details are found in the following subsections.

Fig. 3. The ROM Block holding the challenging problem sequence.
3.1 Sequence multiplexor
The sequence multiplexor gets one sequence at a time. The Logical control issues the signal
to the multiplexor to load the sequence from the ROM and feed the shifter.
3.2 Sequence shifter
The sequence shifter block has the following inputs: clk, reset and the sequence to be shifted.
The shifter outputs an l-sized motif each clock cycle through a windowing approach.

The shifter outputs (n-l+1) motifs for each sequence unless it is interrupted by resetting it.
Our skip Brute Force resets the shifter in one case; when the shifter has generated all the (n-
l+1) l-mers for this sequence. The shifter is reset to be fed with new sequence to generate the
newly suspected motifs (l-mers) from this sequence.

Fig. 4. Sequence Shifter block diagram.
The skip-more algorithm resets the shifter in two cases. The first case is the one previously
explained. The second case happens when the matching unit finds an l to be within the d
allowed mutations. In this case the system resets the shifter as the motif is considered to be
found. Block diagram of Sequence Shifter is shown in Figure 4.
3.3 Motif generator
The set of all 4
l
l-mers starting with AA ... A to TT ...T is not stored in the system. The four
DNA nucleotides {A,C,G,T} are easily encoded into the 2-bit symbols 00,01,10 and 11
respectively. The system locally generates all the possible l-mers by a simple controlled
binary counter of size l bits.
That is, in a system with l=3 we would like to generate AAA, AAC, AAG, AAT, ...,TTT.
According to the encoding mentioned above; we would like to generate a series of 6-bits
each as follows 000000, 000001, 000010, 000011, ..., 111111. The relation between these
encoded bits can be obtained by a simple binary counter of size l bits.
3.4 Matching block
The matching block consists of many sub-blocks; xoring units, an l-bit adder and a
comparison block. The matching block takes two l-sized sequences and compares them. If
the difference between the two sequences is less than or equal to the allowed mutation
(the two sequences have less than or equal to d different nucleotides), it outputs a match
signal.
The matching block uses a series of xoring gates to determine if two l nucletoids are
identical. The l-bit adder is used to count the differences between them. Finally, a
comparison block is used to compare the value obtained from the adder with the d allowed
mutation.
The matching block also outputs the score of the matching process. This score is used by the
logical control to determine the quality of the motif obtained. The Matching block diagram
is shown in Figure 5. Detailed Matching block diagram is shown in Figure 6.


Fig. 5. Matching block diagram.
Our design is meant to be extendible by instantiating more of the matching units. Thus, its
circuit implementation has to be highly optimized. Classical hamming distance circuits start
with an array of XOR gates to determine matching nucleotides, followed by l sequential
adders to compute the required distance. This approach leads to long circuit delays that will
cause the system maximum frequency to drop, degrading the performance.
Our design replaces the sequential adders with a specially designed adders tree. For the (15, 4)
problem, the proposed design shortens the critical path from fifteen 4-bit adders to only four
full adders and two half adders. Figure 7 shows the optimized adder tree.

Fig. 6. Matching block components - xoring units are double the size of the motif.

3.5 Adder tree
The l-bit adder takes a pattern of size l, calculates the number of ones in this pattern and
outputs the count in a log
2
l bits. For l=15, the adder would accept a 15 bit input signal and
ouputs a 4-bit output signal. A 15-bit input signals needs five full adders; this would be
stage 0. Stage 0 outputs 5 sum signals and 5 carry signals. Stage 1 needs 1 full adder and 1
half-adder for the output sum signals and the same for the output carry signals.
Accordingly, stage 2 needs only 4 half adders, stage 3 needs 2 full adder and stage 4 needs 1
half adder. The final stage needs 1 full adder.

Fig. 7. The six stages adder tree - The critical path involves 4 full adders and 2 half adders.
3.6 Logical control
The system is managed by the logical control. Reset signals are issued to the motif generator
and to the sequence shifter to control the flow of the sequences to be compared. As
explained earlier, the logical control issues this signal under certain events. The logical
control outputs the best motif which is determined by the scoring function.
3.7 Multiple matching units
It is clear that scaling up the design by utilizing more matching units in parallel will speed
up the overall performance by the factor of extra units. Slight modifications and some logic
duplication will be introduced for proper functionality and synchronization. The only
limiting factor to the performance boost is the FPGA resources.

Figure 8 shows the block diagram of the skip Brute force running on an FPGA with multiple
matching units. All t sequences are also loaded into an on-chip read-only memory ROM as
the previous architecture. The sequence multiplexor feeds n series of sequence shifter
followed by a matching unit. The matching unit takes its two l-sized sequences one from the
shifter and the other from the logical unit which contains the motif generator. The outputs of
the matching unit in each series are ANDed to determine the value of solution found. The
number of the series of sequence shifter followed by matching unit is equal to n, where n is
the number of the examined sequences. In the previous architecture, the system has to loop
over all the sequences for each generated motif. This corresponds to n.t.4
l
loops. In this
enhanced architecture, the system loops only n . 4
l
.

Fig. 8. Block diagram of the skip Brute Force - running on an FPGA with multiple matching
units.
4. Performance evaluation and results
We tested the performances of Brute Force algorithm and skip Brute Force on synthetic
problem instances generated according to the planted (l,d)-motif model. We followed the
FM model described by Pvzner and Sze [8] to generate synthetic data to test our work. We
produced problem instances as follows:
First, a motif consensus M of length l is chosen by picking l bases at random. Second, t= 20
occurrences of the motif are created by randomly choosing d positions per occurrence
(without replacement) and mutating the base at each chosen position to a different,
randomly chosen base. Third, we construct t background sequences of length n=600 using
n*t bases chosen at random. Finally, we assign each motif occurrence to a random position
in a background sequence, one occurrence per sequence. All random choices are made
uniformly and independently with equal base frequencies.
The skip Brute Force achieves an average speedup of 9.11X. Both Brute Force and skip Brute
Force algorithms were modelled and implemented on MatlabR2006b[15]. All the
experiments ran on an AMD 5500 X2+ processor with 2GB RAM. For fair comparison, it is
reported in literature that the Matlab platform is about 5 to 6 times slower than an
optimized C coded program.
To evaluate the hardware implementation; we need to define the expected number of
matching operations. First, we define the probability to find a random l-mer in a given
sequence with up to d mutations as:


Additionally, we define the expected number of required matching operations to find the
correct implanted motif as:

We then deduce for a problem of size n=600, t=20, the expected matching operations to be as
shown in table 1.

L D Expected Matching
Operations
9 2 7.7699 x 10
7

11 3 1.2388 x 10
9

12 3 4.9428 x 10
9

13 4 1.9750 x 10
10

14 4 7.8813 x 10
10

15 4 3.1464 x 10
11

17 5 5.0170 x 10
12

Table 1. Expected matching operations for different (l,d) problems.
We synthesized our design for multiple matching units (MU). Synthesis results of one, five,
ten and twenty matching units need further analysis. Figure 9 shows the area utilization of
the FPGA. The FPGA utilization increases almost linearly with increasing the number of
MUs.

Fig. 9. FPGA area utilization - increases almost linearly.
The design of multiple MUs inherits parallelization; this means the system critical path
remains the same even after increasing the number of MUs. Unfortunately, the system
maximum frequency decreases with increasing the number of MUs. This is due to the
increased complexity of the FPGA interconnects. Over 80% of transistors inside the FPGA
are dedicated to the programmable routing network as programmable switches and buffers.
The increased complexity of the interconnects leads to FPGA resource starvation.


Fig. 10. Maximum system frequency - decreases due to interconnects complexity.
Furthermore, It is well known that interconnects in FPGA dominate the system performance
and power consumption.
Depending on the architecture, 60% to 80% of the FPGA critical path delay is due to the
routing between logic blocks. Long interconnects exhibit a substantial delay and often lead
to timing violation and require further optimizations. In a recent study [13], it was found
that FPGA interconnects is poorly scaled. Based on the extrapolation of future device
performance, interconnects will become the performance bottleneck, of which the clock rate
will be slowed down to 17 MHz in a 13 nm process. Figure 10 shows degradation in the
maximum frequency of the system with increasing the number of matching units.
We define the system throughput as the number of matching operations per second. Figure
11 shows the curve of the system throughput. The throughput increases by increasing the
number of MUs. The curve tends to be linear but the degradation in the maximum
frequency alters this linearity.

Fig. 11. System throughput - increases almost linearly.
Figure 12 compares the running time of Brute Force, skip Brute Force, skip Brute Force
running on FPGA with one matching unit and with 20 matching units of different challenge
problems. The running time of Brute Force in all challenge problems is the highest. Our skip
Brute Force algorithm running on an FPGA has the best running time.


Fig. 12. Running time of various challenge problems - skip Brute Force running on an FPGA
based architecture with 20 matching units has the fastest running time.
Utilizing one matching unit leads to a speedup by 9800X over pure software running time of
skip Brute Force. It is clear that scaling up the design by utilizing more matching units in
parallel will speed up the overall performance nearly by the factor of extra units. We used
20 matching units and achieved a speed up factor 16.88X over one matching unit.
Thus, applying the skip Brute Force (9.11X) on 20 matching units (16.88X) running on an
FPGA-based architecture (9800X) would offer 1.5MX boosting in the performance.
Figure 13 illustrates these observations.

Fig. 13. Speedup factors of our accelerating designs - Total speedup is 1.5MX.

RTL synthesis and Place and route were accomplished using Quartus tool on the Stratix III
FPGA technology, a product from Altera[14]. The skip Brute Force FPGA design does not
use any of the FPGA memory blocks. The PowerPlay tool showed a total of power
consumption of 400mW.
5. Conclusion and future work
This chapter presents a proof-of-concept parallization of motif finding on FPGA to achieve
high performance at low cost. Among all Motif Finding Algorithms, Brute Force is known to
be the most accurate. This is mainly because it searches the space of all possible motifs. The
major drawback of Brute Force is the intractability of its running time. The algorithm
running time grows exponentially with the length of the motif. This makes the Brute Force
unsuitable for long motifs. The algorithm can not be used to solve the (15,4) challenge
problem in a reasonable time.
In order to find the correct solution for the planted motif problem; we have to over-come
two main problems. We have to be able to identify the motif from background sequences by
applying an exact algorithm such as the Brute Force that guarantees to always find the
correct motif. We also have to overcome its running time and memory complexities through
acceleration by enhancement in the algorithm itself and by hardware implementation. Our
research presented here addresses these two issues.
We presented an enhanced Brute Force algorithm; skip Brute Force, which can predict the
quality of the obtained motif. The algorithm skips those iterations which will lead to a poor
scored motif, thus leads to a better running time. This enhancement guarantees the same
exactness of the Brute Force. Our enhanced algorithm showed a speedup factor of average
9.11X.
The repetitive nature of the algorithm and the locality of the data encourage the use of
FPGAs. Many operations can be done concurrently to enhance the running time. FPGAs
proved to successfully accelerate sequential algorithms minimum by one or two orders of
magnitude. They also have been widely used to accelerate bioinformatics problems such as
Smith-Waterman and BLAST algorithms. This research offers an enhanced Brute Force
algorithm hardware accelerated using Field Programmable Gate Arrays (FPGAs).
We designed an FPGA-based architecture to accelerate our skip Brute Force algorithm. The
core of the skip Brute Force algorithm is its matching unit. Utilizing one matching unit leads
to a speedup by 9800X over pure software running time of skip Brute Force. It is clear that
scaling up the design by utilizing more matching units in parallel will speed up the overall
performance nearly by the factor of extra units. We used 20 matching units and achieved a
speed up factor 16.88X over one matching unit.
Thus, applying the skip Brute Force (9.11X) on 20 matching units (16.88X) running on an
FPGA-based architecture (9800X) would offer 1.5MX boosting in the performance.
Obviously, the real boosting in the performance (9800X) is achieved by introducing FPGA to
the algorithm. It is neither the effect of enhancing the Brute Force algorithm, nor the effect of
applying more matching units.
Many motif finding algorithms achieves better running time on the expense of the motif
accuracy obtained. We succeeded to accelerate the motif finding problem without sacrificing
the accuracy by applying an exact algorithm; skip Brute Force.
Our work can be extended to accelerate other motif finding algorithms that have shown
better performance to solve the motif finding problem. Algorithms such as Projection [4]

and MEME [2] proved to have high accuracy and much better running time. Introducing
these algorithms to hardware acceleration will offer more boosting to its running time.
An embedded processor can be added on the FPGA to run the algorithm on chip. This
approach will eliminate the communication overheads which is the bottleneck in most
hardware-software co-designs.
Furthermore, our approach can be applied to other biological applications. One of the most
important problems in the biological research is the tertiary structure prediction of a protein
using amino acid information. This is particularly important in the context of designer
proteins in the area of drug discovery. Graph analysis of biological networks is also
computationally intensive.
6. References
[1] Rajasekaran, S., Balla, S. and Huang, C.H.: Exact algorithm for planted motif
challenge problems, Proceedings of Asia-Pacific Bioinformatics Conference, 249
259 (2005)
[2] Bailey, T.L., Williams, N., Misleh, C., Li, W.W.: MEME: discovering and analyzing DNA
and protein motifs. Nucleic Acid Research 34, W369W373 (2006)
[3] A. Brazma, I. Jonassen, J. Vilo, and E. Ukkonen, Predicting gene regulatory elements in
silico on a genomic scale, Genome Research 15, 1202-1215(1998)
[4] Buhler, J., Tompa, M.: Finding motifs using random projections. J. Comput. Biol. 9, 225
242 (2002)
[5] Lawrence, C.E., Altschul, S.F., Boguski, M.S., Liu, J.S., Neuwald, A.F., Wootton, J.C.:
Detecting subtle sequence signals: a Gibbs sampling strategy for multiple
alignment. Science 262, 208214 (1993)
[6] E. Eskin and P. Pevzner, Finding composite regulatory patterns in DNA sequences,
Bioinformatics S1, 354-363(2002)
[7] Hertz, G., Stormo, G.: Identifying DNA and protein patterns with statistically signicant
alignments of multiple sequences. Bioinformatics 15(7-8), 563577 (1999)
[8] Pevzner, P., and Sze, S.-H.: Combinatorial approaches to finding subtle signals in DNA
sequences. Proc. 8th Int. Conf. Intelligent Systems for Molecular Biology, 269
78(2000)
[9] Jan SchrOder, Lars Wienbrandt, Gerd Pfei er, and Manfred Schimmler: Massively
Parallelized DNA Motif Search on the Recongurable Hardware Platform
COPACOBANA. PRIB 2008 LNBI 5265, 436-447(2008)
[10] Chen Chen, Bertil Schmidt, Liu Weiguo, and Wolfgang Mller-Wittig: GPU-MEME:
Using Graphics Hardware to Accelerate Motif Finding in DNA Sequences. PRIB
2008 LNBI 5265, 448-459(2008)
[11] Sandve, G.K., Nedland, M., Syrstad, B., Eidsheim, L.A., Abul, O., Drablas, F.:
Accelerating motif discovery: Motif matching on parallel hardware. In: Bcher, P.,
Moret, B.M.E. (eds.) WABI 2006. LNCS (LNBI), vol. 4175, pp. 197206. Springer,
Heidelberg (2006)
[12] Grundy, W.N., Bailey, T.L., Elkan, C.P.: ParaMEME: A parallel implementation and a
web interface for a DNA and protein motif discovery tool. Computer Applications
in the Biological Sciences (CABIOS) 12, 303310 (1996)
[13] Terrence Mak The Future Looks Gloomy for FPGA Interconnects Technical Report
Series NCL-EECE-MSD-TR-2009-145, 2009.

[14] Altera Inc., http://www.altera.com/
[15] Matlab Product Family. http://www.mathworks.com.
6
A Pattern Search Method for Discovering
Conserved Motifs in Bioactive Peptide Families
Feng Liu
1
, Liliane Schoofs
2
, Geert Baggerman
2
,
Geert Wets
1
and Marleen Lindemans
2
1
Data Analysis & Modeling Group, Transportation Research Institute, Hasselt University
2
Functional Genomics and Proteomics, Department of Biology, K.U. Leuven
Belgium
1. Introduction
Bioactive peptides play critical roles in regulating most biological processes in animals, and
they have considerable biological, medical and industrial importance. Peptides belonging to
the same family are often characterized by a typical short sequence motif (pattern) that is
highly functionally preserved among the family members. In this chapter, we design a
pattern search method to facilitate the detection of such conserved motifs. First, all known
bioactive peptides annotated in Uniprot are collected and classified, and the program Pratt
is used to search these unaligned peptide sequences in each family for conserved patterns.
The obtained patterns are then refined by taking into account the information on amino
acids at important functional sites collected from literature, and are further tested by
scanning them against all the Uniprot proteins. The diagnostic power of the patterns is
demonstrated by the fact that, while the false positive is kept to zero to ensure that the
signatures are exclusive to peptides and their precursors, nearly 94% of all known peptide
family members accommodate one or several of the identified patterns.
In total, we brought to light 155 novel peptide patterns in addition to the 56 established ones
in the PROSITE database. All the patterns represent 110 peptide families; among which 55
are not characterized by PROSITE and 12 are also dismissed by other existing motif
databases, such as Pfam. Using the newly uncovered peptide patterns as a search tool, we
predicted 95 hypothetical proteins as putative peptides or peptide precursors.
2. Problem statement and background
Whole genome sequencing projects have made available immense sequence data at a pace
that far supersedes their rate of annotation. As a result, out of 1.7 million protein sequences,
which are currently available for all the completely sequenced metazoan genomes, nearly
15% could not be assigned to any putative function. Although several tools/algorithms are
available to contribute towards the putative functional assignments of the proteins, yet large
numbers of proteins remain un-elucidated. In most cases this is due to the low degrees of
sequence similarities with known proteins; alternatively, the existing similarities can be
confined to only very small part(s) of the entire protein. The latter is especially true for
precursor proteins coding for bioactive peptides. Consequently, there is still a need for


122
bioinformatic tools to predict the function of the enormously large number of the unknown
protein sequences.
Bioactive peptides occur in the whole animal kingdom, from the least evolved phyla to the
highest vertebrates (Filipsson et al., 2001; Masashi et al., 2001). They play key roles as
signaling molecules in many, if not all physiological processes, for instance as a peptidergic
neurotransmitter or neurohormone, as a peptidergic toxin, or as a growth factor (Boonen et
al., 2007; Boonen et al., 2010). They are synthesized in the cell in the form of large
preproproteins (precursors), which are a special class of proteins as they undergo extensive
post-translational processing prior to producing final mature bioactive peptides (Schoofs &
Baggerman, 2003). Peptides and their precursors that are structurally and functionally
related have been classified into peptide families; each family of proteins is assumed to be
derived from a common ancestor (Husson et al., 2009). During the evolutionary process, the
protein sequences may have much diverged, but the essential amino acids involved in the
biologically important activities are still present. These conserved amino acids along with
their particular sequential order form the functional foundation and represent the motif
(pattern) of a peptide family.
However, over the course of natural adaptation, different peptide families have diverged at
different rates. While for some peptide families, the similarity extends over a much longer
region even over the entire peptide precursor sequences; for many others, a short highly
conserved motif is responsible for the function of the precursor proteins throughout the
family members, and the sequence fragments outside the conserved regions often display no
significant similarities (Baggerman et al., 2005). The latter conserved sequence characteristics
can be further exposed by many short but biologically important functional peptides
released from known large precursors as annotated in Uniprot, such as the 3-amino-acid
thyroliberin peptide QHPamide (Vandenborne et al., 2005) and 4-amino-acid
neuropeptides FMRFamide (Baggerman et al., 2002). For some mature peptides, the
precursor proteins (genes) are unknown, such as the 2-amino-acid neuropeptide GWamide
(P83570) from Sepia officinalis (Henry et al., 1997) and the human growth-modulating
peptide GHK (P01157) (Schlesinger et al., 1977). The existence of numerous short bioactive
peptides within the precursor proteins implies that only a very small conserved peptide
motif may be a biologically important functional portion of the precursors.
Due to the fact that only short sequence regions are conserved, peptides or their precursors
are sometimes not identified by existing sequence alignment algorithms e.g. BLAST or by
motif search methods. While BLAST programs (Altschul et al., 1997) are very suitable to
scan databases for homologous proteins, they are far less efficient at finding similarities to
short conserved regions which can be only a few amino acids in length, when the whole
genome sequence is scanned. For large precursors which are usually a few hundred amino
acids in length and for which the biologically conserved regions are limited, the important
domains are often masked by long randomly unrelated sequence regions. This is because for
any two random large protein sequences, BLAST usually can find a relative long local
alignment, at least longer than the short conserved peptide motif, and BLAST tends to
assign a higher score to a longer alignment (Durbin et al., 1998). In addition, if a pair of
homologues involves a short independent peptide molecule, which may be either an
unknown peptide sequence as query or a known mature peptide as target from a protein
database, it is difficult for BLAST to detect the pair of homologues, because the involvement
of a short sequence makes the pairwise sequence alignment less likely to obtain a significant
BLAST score (e.g., e-value < 0.01).

A Pattern Search Method for Discovering Conserved Motifs in Bioactive Peptide Families

123
Like BLAST, motif search methods are important tools to search for a protein in a database,
nevertheless, they are also limited to detect all members from a characterized peptide
family. Most of the motifs in the existing databases, e.g. PROSITE (Hulo et al., 2004) and
Pfam (Finn et al, 2010), cover the entire precursor sequences or sequence domains which are
much longer than the conserved bioactive peptide regions. Therefore, the database motifs
show their weakness when they are used to detect short mature peptides for which the
precursors are unknown and the information on the sequences outside the peptide regions
is thus missing. In addition, the construction of these motifs requires a good multiple
protein sequence alignment in order to produce an accurate signature. This works well
when the sequences are easy to align. However, for some peptide families for which the
conserved regions are very short and the bulk of peptide precursor sequences is not very
well preserved, the multiple alignment is very difficult to obtain or evaluate. The overall
precursor protein sequence identity, especially in distantly related homologues, may be too
low for an accurate alignment. In some cases, the short conserved regions are repeated
within a precursor, making it even more challenging to build a unique alignment that truly
reflects the evolutionary relationship.
In this chapter, we have followed an alternative approach, taking unaligned sequences as a
starting point. We then used a pattern search program to look for conserved patterns. We
first collected all currently annotated peptides and peptide precursor proteins in Metazoa
through a search in Uniprot and classified them into peptide families. Next, we extracted
peptide sequences in each family and used the program Pratt to search the sequences for
representative patterns. Such patterns consist of highly conserved positions that can be
separated by fixed or variable spacing. The patterns are then refined by incorporating the
information that is available in literature on the important amino acids contained within the
biologically active site(s) of the peptides. The specificity of the generated patterns are further
verified by scanning them against Uniprot in order to ascertain that proteins picked up by
the patterns are either annotated as peptides or peptide precursor proteins or have an
unknown function.
3. Data collection
3.1 Peptide precursor collection and classification
A protein was collected into a peptide-precursor database if it is annotated in the Uniprot
protein database (release 6.6) consisting of Swiss-Prot (release 48.6) and TrEMBL (release
31.6) with one of the following keywords: hormone, antimicrobial, toxin. The hormone
includes bombesin, bradykinin, cytokine, glucagon, growth factor, hormone, hypotensive
agent, insulin, neuropeptide, neurotransmitter, opioid peptide, pyrokinin, tachykinin,
thyroid hormone, vasoactive, vasoconstrictor and vasodilator (the definition of the
keywords can be referred to in this database). The antimicrobial consists of antibiotic,
antiviral defense, defensin and fungicide; while the toxin includes naturally produced and
secreted poisonous proteins that damage or kill other cells. However, when the protein is
also characterized by non-peptide keywords, such as receptor, signal-anchor,
transmembrane, binding protein, DNA binding, nuclear protein, transport, collagen,
enzyme or words ending in ase (excluding disease), it is excluded, in order to avoid the
selection of proteins which are not peptides or peptide precursors.
Stand-alone PSI-BLAST (ftp://ftp.ncbi.nih.gov/blast/executables/) is then used to align all
the assembled sequences with all the Uniprot proteins except the ones which are already in


124
the peptide-precursor database. Based on the conserved sequence characteristics of peptide
families, the score matrix PAM30 is used and the word size is set to 2, allowing for the
search for short but strong similarities. The proteins, which show significant similarities (e-
value <0.01) with the known peptides or precursors, are retained. The obtained list is then
checked manually in terms of the proteins cellular location, molecular function and
biological process as stated by GO (gene ontology) terms or in literature. As a result, 1345
more proteins which have as yet not been annotated in Uniprot are added to the peptide-
precursor database.
Proteins collected in this database are automatically classified into peptide families if their
family classification information is available in Uniprot that is based on a significant match
to an existing motif or based on sequence similarities. Otherwise, proteins that display
sequence similarities with a significant BLAST score, are clustered into the same family. A
protein can also be assigned to a particular family based on its molecular function described
in literature.
3.2 In silicon extraction of peptides
From each precursor protein in a peptide family, the bioactive peptide sequences are
extracted in silicon from the beginning and ending positions of the subsequences that are
annotated as peptide or chain in feature line in the corresponding protein file in Uniprot.
The conserved basic cleavage sites flanking the peptides, which contribute to the
endoproteolytic cleavage process of the peptides from their precursors, such as the
monobasic site (G)R or (G)K, the dibasic sites (G)KR, (G)RR, (G)KK or (G)RK, or a
combination of consecutive K or R, are also withdrawn along with the subsequences (Liu &
Wets, 2005; Rouille et al., 1995).
Entries in the family that only constitute the peptide sequence, i.e. in those cases where the
precursor is unknown, are also retained. Proteins less than 200aa (amino acids) in length,
which contain an N-terminal signal peptide and for which no mature peptides have as yet
been identified, presumably contain a single peptide and are therefore also deposited after
in silicon removal of the N-terminal signal peptide. According to the statistics on all
annotated bioactive peptide sequences in Uniprot, 97% are no longer than the 200aa
threshold value. The presence of a signal peptide is assumed when it is indicated in Uniprot;
in other cases, it is forecasted by the signal peptide prediction program signalP
(http://www.cbs.dtu.dk/services/SignalP/).
In total, 110 datasets of peptide families are formed with each including at least 10 peptide
sequences. All the extracted peptide sequences in each of the families were scanned
independently for patterns conserved in the corresponding family.
4. Method
Different software available on the internet provides users the tools to search for patterns
conserved in a set of unaligned protein sequences. Pratt (http://www.ebi.ac.uk/pratt/#)
(Jonassen et al., 1995) is a flexible pattern search tool in the number of parameters that can
be controlled by users. It allows searching for patterns of conserved positions with limited
variable length spacing, which is important because even in well-conserved peptide regions,
variable loop sizes can occur. Pratt is run on each of the peptide family datasets, and the
searching parameters are set based on maximum pattern length and pattern flexibilities
found in the existing peptide patterns in PROSITE.


125
For each Pratt run which starts with the minimum percentage of sequences to match the
pattern (the parameter C%) equal to 90%, the most significant pattern, which is the one with
the highest fitness in the Pratt output list, is retained. The obtained pattern is then refined by
integrating the information on the important functional sites in the matched peptide
sequences depicted in literature. The amino acids occurring at these sites are added to the
pattern if they are absent at the corresponding sites in the pattern.
The pattern is further verified by scanning it against all the Uniprot proteins using the
ScanProsite tool (http://www.expasy.org/tools/scanprosite/). Two possible cases occur:
(1) If the pattern is not contained in any known non-peptide protein, it is retained as a
conserved peptide pattern. (2) Otherwise, if the pattern is matched by both peptide and non-
peptide proteins (further referred to as true and false positive hits, respectively), it is
subsequently processed as follows. (2a)If the pattern does not include any wildcard region
where any amino acid is accepted, the positions where the pattern is located in all matching
protein sequences are checked. If the pattern exclusively occurs at the N- or C-terminus of
the true positive hits, or if the peptide proteins are all small molecules, the pattern is
retained with a constraint (< or >) imposed at the N- or C-terminus of the pattern to limit
the maximum distance between the conserved pattern region and the N- or C-terminus of
the peptide or precursor protein. If the pattern with such a restriction cannot distinguish the
true positives from the false ones, the pattern is eliminated. (2b)Or, if the pattern has
wildcard regions, the sequence fragments corresponding to the pattern in all the matching
sequences are extracted and aligned. If the two groups of amino acids in a wildcard region X
in this alignment have different physicochemical properties between the true and the false
positive hits, the region X is replaced by the group of amino acids distinctively occurring in
the true positive proteins. In the other case, when the two groups of amino acids share
identical physicochemical properties, the pattern is discarded. The amino acid symbol sets:
DE, KRH, NQ, ST, ILV, FWY, AG, C, M and P, which are classified based on the
physiochemical nature of the side groups (Smith & Smith), are used.
If a conserved pattern cannot be obtained, the parameter C% is reduced by 10%, and Pratt is
re-run against the same dataset. As the percentage of sequences to match the pattern
decreases, a pattern which is usually longer and contains more sites than the previously one
is shown up and processed by similar refinement and verification. The procedure is
repeated until a pattern, which represents the majority of a group of related peptide
sequences and rules out any known non-peptide proteins, is discovered.
Once a conserved pattern is identified in the peptide family dataset, the program ps-scan
(ftp://ftp.expasy.org/databases/prosite/tools/ps_scan/sources/) is run locally on the
pattern against this dataset. The sequence regions which match the pattern are removed
from the original peptides. Each of the two remaining parts of the peptide sequences at their
N- and C-terminus is left to form an independent sequence if it is not less than 4aa in length,
given the assumption that the minimum length of the peptide pattern we search for is not
less than this value. Thus, a reduced dataset is created including not only the peptides
which are not covered by the identified pattern, but also the remaining sequences of the
original peptides that match the pattern. This methodology is based on the fact that a
peptide precursor protein may contain several conserved regions, and that our extracted
peptide sequences include long peptide chains which may contain a few shorter, unrelated,
bioactive peptides. The reduced peptide family dataset is then scanned by Pratt to discover
the next pattern. The search procedure is repeated until the parameter C% is less than 50%.


126
This means that the remaining dataset contains no more patterns representing the majority
of the sequences.
Fig. 1 represents the scheme of the described pattern searching procedure which is aimed to
examine short bioactive peptide sequences rather than their large precursor molecules, and
to take into account not only the biologically functional sites of each individual peptide
discussed in literature, but also the general information which is extracted by the
computational tool Pratt from all related peptides in a family.
5. Results
5.1 PeptideMotif database
We have built a peptide-precursor database consisting of 11,688 peptides and precursor
proteins originated from 1420 metazoan organisms; of which 11,437 proteins (98%) are
categorized into 110 distinctive peptide families. Based on bioactive peptide sequences
drawn from the peptide families, we uncovered in total 211 conserved patterns which are
assembled into the peptide motif database PeptideMotif.
All the patterns range between 4 and 52 amino acids (column) in length with 78 (37%) no
longer than 10aa. While each of the patterns covers most of the peptides or precursors
belonging to the corresponding family, the false positives are kept to zero because it is
guaranteed by the criterion that a known protein matching the pattern is indeed a peptide or
precursor protein from this family.
5.2 Comparison with the other motif databases
The PROSITE database (http://ca.expasy.org/prosite) is a motif database of protein families
and domains. It consists of biologically significant sites, patterns and profiles that help to
reliably identify to which known protein family (if any) a new sequence belongs. Its 19.9
release contains 56 entries (patterns) describing 55 peptide families in Metazoa (the omega-
atracotoxin family has two patterns) belonging to categories of cytokines and growth
factors, hormones and active peptides, and toxins. All the 55 families are also covered by
patterns in the PeptideMotif database, and these peptide patterns (Table 1) share the
similar length to their PROSITE counterparts. However, in terms of conserved sequence
characteristics revealed in both database motifs, more amino acids are imposed at the
conserved sites or wildcard regions in the PeptideMotif patterns. This is due to the fact that
the identified peptide patterns are not only trained by running them against the Swiss-Prot
protein database which is also used as the test dataset by PROSITE, but also against the
TrEMBL database, in which many proteins are also annotated by keywords or literature. In
addition, for 25 of the 56 families, we have found 34 additional novel patterns and they are
marked as new in Table 1.
The remaining 121 PeptideMotif patterns presented in Table 2 allow the identification of 55
peptide families that are untouched by PROSITE signatures; they cover 3866 bioactive
peptide sequences cleaved from 3572 precursors. Among the patterns, 28 representing 12
families are also not characterized by any other motif database, such as Pfam (Bateman et
al., 2004) and CDD (Marchler-Bauer et al., 2005). The sequence reminiscence for these
families is short and often occurs repeatedly within a same precursor protein. The sequences
outside the conserved region are not well preserved, and thus a probability model based on
protein sequence alignments cannot efficiently characterize such peptide families.


127

Fig. 1. Procedure for searching patterns in peptide sequences.
Note: The parameters are set as follows: the maximum pattern length (PL) is 52, the
maximum length of a wildcard (PX) is 15, the maximum number of flexible wildcards (FN)
is 3, the maximum flexibility of a flexible wild card (FL) is 8, the upper limit on the product
of flexibilities for a pattern (FP) is 48, the minimum percentage of sequences to match the
pattern (C%) is 90, 80, 70, 60 and 50%, respectively, and all other parameters are at default.
Yes
Yes No
No No
Yes Yes
No
No
Yes
Begin
A peptide family dataset
Run Pratt and select the
most significant pattern
Refine by literature
Run ScanProsite to the
pattern against Uniprot
A conserved pattern
is obtained
Run ps-scan locally
against the peptide
family dataset
Remove the matched
sequence parts, a
reduced dataset is
created
C% is reduced by
10%
Align all matched
sequences in the
peptide and non-
peptide proteins
New constraints are
imposed at the wild
regions in the pattern
A constraint is
imposed at the N- or C-
terminus of the pattern
Minimum Percentage (C%)=90%
If the letters
at X have
different
properties
between
peptides and
non-
peptides
If the pattern
is at the
terminus of
precursors,
or if the
precursors
are small
molecules
If the pattern
has wild
regions
If C% >=
50%?
If the pattern
is contained
in non-
peptide
proteins
End


128
Cytokines and growth factors
(1) Granulins; (1) C-x-D-x(2)-H-C-C-{LIVM}-x(4)-C; {42, 241, 2}; {Q616A1, Q7JKP2, Q9U362}
(2)HBGF/FGF; (1) G-x-[LIVM]-{AGNP}-[STAGP]-{AGC}-{C}-x-{KRHNDE}-{WPC}-x-
[STAGDENKRHQ](0,1)-[AGST](0,1)-[DENA]-C-{QP}-[FYLIVM]-{C}-[EQH]-x-{P}-{C}-{LIVM}-
[DENKRHL]-{PLIVMDE}-[YHF]; (2) [GR]-[LIVM]-[LIVM]-{CWPDE}-[LIVM]-{PST}-{QLIVM}-x-
[KRDEVIAGQFYNCS]-[STAGLMHQ]-{CP}-{AGDEN}-[FY]-[LIVM]-[AGSC]-[MLIV]-[NSTDEK]-
[GAKRSTNDEQ]-[EDNKRHSTQA]-G(new); (3) G-S-[RHKQ]-[LIVM]-{CWPDE}-[LIVM]-{PST}-
{QLIVM}-x-[KRDEVIAGQFYNCS]-[STAGLMHQ]-{CP}-{AGDEN}-[FY]-[LIVM]-[AGSC]-[MLIV]-
[NSTDEK]-[GAKRSTNDEQ]-[EDNKRHSTQA]-G (new); {300,530,44}
(3) PTN/MK heparin-binding; (1) S-[DE]-C-x-[DE]-W-x-W-x(2)-C-x-P-x-[SN]-x-D-C-G-[LIVMA]-G-
x-R-E-G (identical); (2) C-[KR]-[YF]-x-[KRFY]-x(2)-W-[AGST]-x-C-[DENST] (new); {51, 84, 1}
(4) Nerve growth factor; (1) [GSRAED]-[CR]-[KRLIVM]-G-[LIVAT]-[DE]-{C}-x(2)-[YW]-{P}-S-x-[CR];
(2) [SAP]-[LIVA]-C-[DEY]-[SAG]-{WM}-[STDENC]-x-W-[VE]-[AGSTNI] (new); {321, 471, 12}
(5)Platelet-derived growth factor (PDGF); (1) P-[PSRAKQGL]-C-[LIVMFYAGST]-x(3)-[RQ]-C-
[AGSTMLIVN]-G-S(0,1)-[CN]-C; {158, 158, 23}
(6)Small cytokines C-x-C; (1) C-x-C-{CFYW}-{CW}-x(3)-{P}-x(2)-{C}(8)-x(5,8)-C-x(2,3)-[EQMA]-
[LIVMTE]-[LIVMF]-x(9,14)-C-[LIVMRK]-[DENH]; {206, 206, 18}; { Q6DUZ6, Q6GLX8, Q4T8B9}
(7) Small cytokines (intercrine/chemokine) C-C; (1) C-C-[LIVMFYSTQRKHDE]-{P}(2)-{CDE}-{C}(7)-
x(2,5)-{P}-[FYWAC]-{C}(2)-x(3,6)-C-{KM}-{C}(1,3)-[SAG]-[LIVMTS]-[LIVMRTDE]-[FYLIVDE]-
{C}(7,10)-C-[STAGVILM]; {234, 234,27}; { Q3ZBN3, Q32L58}
(8)TGF-beta; (1) [WFYSTKRHL]-[LIVM]-[LIVMKRHF]-{CPNL}-P-{FY}-{PCW}-[FYILVA]-{C}-
{QCWKRH}-{PA}-{PAGC}-C-{C}-[GE]-{C}-C; {766, 766, 59}
(9)interferon alpha, beta and delta; (1) [FYH]-[FY]-{CP}-[GNRKCDSTI]-[LIVM]-{W}-{AGC}-
[KRN](0,1)-[FYLVIMN]-L-{PAG}-{C}-{PST}-{PFYW}-[FYHDEN]-x-{QY}-[CYQE]-[AT]-W; (2) L-
{QKR}-x(0,4)-[GAEDVI]-[LVI]-[QHNDEFY]-[RQ]-[QH]-[LMIV]-[DENQVSTR]-x-L-[DENKRQ]-x-C-
[LIVMKRQG] (new); {272, 442, 29}
(10)Granulocyte-macrophage colony-stimulating factor; (1) C-P-[LP]-T-{ST}-E-x-{QLIVMT}-C; {25,
25, 1}; {Q4G094}
(11) Interleukin-1; (1) [LIVSTNDEFH]-[YESTMVIR]-[LFC]-{AGCFYL}-[SA]-[ASLV]-{CFY}-
[CFYWH]-[PKRST]-{FYLC}-[WHLIVM]-[FYL]-[LI]-[SCA]-[TSVG]-x(6)-[PKRHCLIVMT]-x(0,2)-
[LIVM]-[AGSTCVINDE]; {128, 128, 24}
(12) Interleukin_2; (1) [ST]-E-[LF]-x(2)-L-x-C-L-x-[EDN]-E-L; {74, 74, 14}
(13) Interleukin_4_13; (1) [LI]-x-E-[LIVM](2)-{Q}(4)-x(0,1)-[LIVM]-[TL]-x(5,7)-C-x(2)-[LMIVST]-x-
[IV]-x-[DNS]-[LIVMA]; (2) [KREV]-N-[STA]-[STED]-[DEAG]-{C}(3,4)-C-[RKT]-[AV]-x(11,17)-C
(new); {73, 119, 4}
(14) Interleukin_6; (1) C-x(9)-C-[FYLIVM]-x(5)-G-L-x(2)-[FY]-x(3)-L; {69, 69, 8}
(15) Interleukin_7_9; (1) N-[DAT]-[LAPS]-[SCT]-F-L-K-{AGDE}-L-L; {20, 20, 2}
(16) Interleukin_10; (1)[KQSN]-{C}(4)-C-[QYCH]-x(4)-[LIVM](2)-x-[FL]-[FYT]-[LMVRT]-x-[DERST]-
[IV]-[LMF]; {75,75,12}
(17) LIF / OSM; (1) [PSTA]-x(4)-F-[NQ]-x-K-x(3)-[CG]-x-[LF]-L-x(2)-Y-[HK] ; {24, 24, 4}
(18) Osteopontin; (1) P-x(1,5)-[KQ]-x-[TA]-x(2)-[GA]-S-S-E-E-K; {27, 27, 0}
Hormones
(19) Adipokinetic (1) [AGC]-Q-[LVI]-[NT]-[FY]-[ST]-[PASTKR]-[AGWSDEN]-W-[AGNDEST]; (2)
<Q-[LVI]-[NT]-[FY]-[ST]-[PASTKR]-[AGWSDEN]-W-[AGNDEST>] (new); {45, 45, 0} {Q5TTQ9}
(20) Bombesin-like peptides (1) [HLIVMQ]-W-A-[STIVRK]-G-[SH]-[LF]-M; {42, 42, 1}
(21) Calcitonin/CGRP/IAPP (1) [KR]-R-x(0,1)-C-[SAGDNT]-[STNG]-x(0,1)-[STAGVIL]-[TS]-C-
[VMALI]-x(3)-[LYF]-x(3)-[LYFVI]; (2) <x(0,1)-C-[SAGDNT]-[STNG]-x(0,1)-[STAGVIL]-[TS]-C-
[VMALI]-x(3)-[LYF]-x(3)-[LYFVI] (new); {83, 84, 7}


129
(22) Corticotropin-releasing factor (1) [KR]-R-x(0,28)-[PQASLVIG]-[STPI]-[LIVM]-S-[LIVM]-x-
[LIVMNAG]-[PST]-[LIVMFT]-x-[LIVM]-[LM]-[RN]-x(2)-[LIVMWF]; (2) <x(0,8)-[PQASLVIG]-[STPI]-
[LIVM]-S-[LIVM]-x-[LIVMNAG]-[PST]-[LIVMFT]-x-[LIVM]-[LM]-[RN]-x(2)-[LIVMWF] (new); (3) T-
R-[PQASLVIG]-[STPI]-[LIVM]-S-[LIVM]-x-[LIVMNAG]-[PST]-[LIVMFT]-x-[LIVM]-[LM]-[RN]-x(2)-
[LIVMWF] (new); {64, 64, 9}; {Q4RWF4}
(23) Arthropod CHH/MIH/GIH neurohormones (1) [LIVM]-{C}-x(2)-C-[KR]-{FY}-[DENGRKHQ]-C-
[FY]-{C}-{AGKRC}-{C}(2)-[FYILVM]-{C}-{CP}-C; {135, 135, 5} {Q23247}
(24) Erythropoietin/thrombopoeitin (1) P-x(4)-C-D-x-R-[LIVM](2)-x-[KRH]-x(14)-C; {34, 34, 8, 0}
(25)Granins (1){DEF}-[DE]-[SN]-L-[SAN]-[AD]-[LIMVKR]-[DE]-[AGLSTQ]-E-L; (2) [LIVM]-x-
[KHR]-C-[LIVM](2)-[ED]-[LIVM](2)-x(5)-[KRH]-[STP]-x(3)-[PST]-x(4)-C (new); (3) K-R-[STAG]-
[NDEST]-[ED]-x(2)-[DE]-[DEGA]-[QKR]-Y-[AGST]-P-Q (new); {63, 96, 5}; {Q86T07, Q4RYY8,
Q566G8}
(26) Galanin (1) G-W-[ST]-L-N-[ST]-[AG]-[AG]-[FY]-[LIVM]-[LIVM]-G-P; (2) <L-N-[ST]-[AG]-[AG]-
[FY]-[LIVM]-[LIVM]-G-P (new); {31, 31, 1}
(27) Gastrin/cholecystokinin (1) [FY]-x(0,2)-[GADN]-[AS](0,1)-[WH]-[MFLIV]-[DR]-F-G-[KR]-[RS];
(2) Y-x(0,2)-[GA]-[AS](0,1)-[WH]-[MFL]-[DR]-F> (new); {88, 102, 4}
(28)Glucagon/GIP/secretin/VIP (1) [YH]-[STAIVGD]-[DENQ]-[AGF]-[LIVMSTE]-[FY]-
{QLPAGDEKR}-[DENSTAK]-[DENSTA]-[LIVMFYG]-[RKSTDEN]-x(3)-{P}-{P}-x(2)-[AGSTLIVMQ]-
[KREQL]-[KRDENQL]-[LVFYWG]-[LIVQ]; {202, 305, 8}
(29) Glycoprotein hormones alpha chain (1) C-x-G-C-C-[FY]-S-x-A-[FY]-P-T-P; {109, 109, 4}
(30) Glycoprotein hormones beta chain (1)C-{C}(2)-[CW]-{C}(7,9)-C-[STAGMLIVED]-G-[HFYLRS]-
C-{C}-[STA]; (2) <x(0,8)-C-[STAGMDEVLI]-G-[HFYL]-C-{CKRH}-[ST] (new); (3) <x-[CW]-{C}(7,9)-C-
[STAGDEVLIM]-G-[HFYL]-C-{C}-[ST] (new); {341, 341, 13}
(31) Gonadotropin-releasing hormones (1) Q-[HY]-[FYW]-S-x(4)-P-G-G-[KR]-R; (2) Q-[HY]-[FYW]-
S-x(4)-P-G> (new); {178, 188, 4}
(32) Insulin (1){C}(2)-[IVLMPSTAFYR]-{CNE}-x-{C}-C-C-{CPM}-{P}-{CHW}-C-[STDNEKIGQ]-{C}(2)-
{CPAG}-[LIVMFSQ]-{CD}-{CPW}-{CHDEP}-C; (2) <x(0,205)-C-G-{FYILVMQW}-{CWPSTLIVM}-
[LIVFY]-[VILMASTPH]-{AGHCFYPQW}-{CPQSW}-[LIVMRKHQWF]-{CNP}-{WCQP}-[LVIMATC]-
C-{LM}-x(0,204)> (new);{507, 877, 52} {Q32L79, Q621L6, Q61VN2, Q61GN7, Q4T1R8}
(33) Natriuretic peptides (1) C-F-G-x(3)-[DEA]-[RH]-I-x(3)-[ST]-x(2)-G-C; {155, 155,10}
(34) Neurohypophysial hormones (1) C-[LIFY]-[LIFYV]-x-N-C-P-x-G; (2) C-x(2,6)-[CW]-G-x(4,6)-C-
[FYAGLIVM]-x(3)-[LIVFY]-C-C (new); {112, 259, 4}
(35) Neuromedin U and S (1) [FY]-[LIVMF]-[FY]-R-P-R-N-G-[KR]; (2) [FY]-[LIVMF]-[FY]-R-P-R-N>
(new); {24, 24, 3}
(36) Pancreatic (1) [FY]-x(2)-{LIVM}-[LIVM]-x(2)-[YK]-x(3)-[LIVMFYRHK]-x-R-[PQVH]-R-[YF]-
[GD]-[KR]-[RS]; (2) [FY]-x(3)-[LIVM]-x(2)-[YK]-x(3)-[LIVMFYRHK]-x-R-[PQVH]-R-[YF]-x(0,1)>
(new); {118, 118, 7}
(37) Parathyroid hormone (1) [KR]-R-x-[VI]-[STAGFYN]-[EH]-x-Q-x(2)-H-[DEN]-x-[GR]; {54, 54, 3}
(38) Pyrokinins (1) [AGHNQDEST]-{FYST}-[PQVIWFYED]-[FY]-[AGST]-P-R-[LI]-G-[KR]-R; (2)
[AGHNQDEST]-{FYST}-[PQVIWFYED]-[FY]-[AGST]-P-R-[LI]> (new); {72, 89, 4} {Q7PTL2, Q5TV14}
(39) Somatotropin (1) C-{KRAG}-[STNRAC]-x(2)-[LIVMFYSRNW]-x-[LIVMSTAGY]-P-x(2)-{FYW}-
x(2)-[TALIVMSHN]-x(7)-[LIVMFYP]-x(2)-{QHKR}-{KRHP}-{NW}-x-[LIVMFYR]-[LIVMSTC]-x-
[STACVLMIG]-W; (2) C-[LIVMFG]-x-[KHRSNDEQVI]-[DEN]-{CNDEPQ}-{AGLMVI}-[KRMT]-
{DENKRHPQ}-x-[STNALIVMF]-[FYLIVMKS]-[LIMVT]-x-{NDEKRH}-[LIVMATE]-[KRNEQTA]-C
(new); (3) [ED]-K-L-L-[DE]-R-[VIA]-[IV]-x-H-[AT]-E-L (new); (4) C-F-[KRH](2)-[DEN]-[LIVMAG]-
[HKR](2)-[LIVM]-[DEQ]-[ST]-[FYLIVM]-x(0,1)> (new); {633, 1093, 45}
(40) Tachykinin (1) [AGSTQKRFY]-[SF]-[IVFYTHQ]-G-[LVIM]-M-G-[KR]-[RS]; (2) [AGSTQKRFY]-
F-[IVLMFYSHQ]-G-[LVIMS]-R-G-K-R (new); (3) <x(0,9)-F-[IVLMFYTHQ]-G-[LVIMSTAG]-[RM]>
(new); {104, 124,6}


130
(41)Urotensin II (1) C-F-W-K-Y-C (identical); {30, 30,1}
(42) Endothelin (1) C-{C}-C-{C}(4)-D-{C}(2)-C-{C}(2)-[FY]-C; {50, 104, 2}
(43) Agouti (1) C-{C}(6)-C-{C}(6)-C-C-{C}(2)-C-{C}(2)-C-{C}-C-{C}(5,6)-C-{C}-C-{C}(6,9)-C; (2) C-
{C}(6)-C-{C}(6)-C-C-{C}(2)-C-{C}(2)-C-{C}-C-{C}(5,6)-C-{C}-C-{C}(0,8)> (new); (3) C-{C}(6)-C-{C}(6)-C-
C-{C}(2)-C-{C}(2)-C> (new); (4) C-{C}(6)-C-{C}(6)-C-C-{C}(2)-C-{C}(2)-C-{C}-C-{C}(5,6)-C(0,1)> (new);
{37, 37, 7}
Antimicrobial
(44) Cecropin (1) W-[KDN]-{QNDEGAKRW}-[FYGA]-K-[KRE]-[LIVM]-E-[RKHAGN]-x-[AGVI]; (2)
[GS]-[WRKHG]-[LIVMST]-[KRST]-K-{QNDEGAKRW}-[FYGA]-K-[KRED]-[LIVM]-E-[RKHAGN]-x-
[AGVI] (new); {96, 96, 3} {Q5TWE5}
(45) Mammalian defensins (1) C-{C}-C-{C}(3,5)-C-{C}(6)-{CP}-[GARKSTW]-x-[SC]-{C}(6,10)-C-C; (2)
C-[PR]-x-C-x(2,5)-C-x(2)-C-[PQ]-x-C-[PQ]-x-C (new); {119, 145, 5}
(46) Arthropod defensins (1) [CG]-x(0,1)-{C}-{CQ}-[HNSEDRY]-C-x(3)-{C}(0,1)-[GR]-{A}-x-
[GRQAY]-[GAL]-x-C-{FY}-x(3,4)-C-{C}-C; (2) [CG]-x(0,1)-{C}(2)-[HNSEDRY]-C-x(3)-{C}(0,1)-[GR]-
{A}-x-[GRQAY]-[GAL]-x-C-{FY}-x(6)-C-{C}-C (new); {103, 105, 7}; {Q6XD83}
(47) Cathelicidins (1) Y-{LIVM}-[EDQN]-[AVI]-[LMVI]-{HKRG}-[RKHQ]-A-[LIVMA]-[DQGEN]-x-
[LIVMFY]-N-[DEQ]; {58, 58, 0}
Toxin
(48) Snake toxins (1) C-{CKRPL}-x(0,2)-C-[PRTFG]- {C}(5)-x(0,6)-C-C-{P}-x-[PDEN]-x-C-[NDEY];
{352, 352, 20}
(49) Myotoxins (1) K-x-C-H-x-K-x(2)-H-C-x(2)-K-x(3)-C-x(8)-K-x(2)-C; {15, 15, 0}
(50) Scorpion short toxin 1 (1) C-{C}(4,5)-C-{PC}-{CQ}-{C}-C-x(3)-{C}-{CPWA}-x(1,4)-[GASEDN]-
[KRAVISNDE]-C-[VIMQTDK]-[NG]-x(1,2)-{P}-C-[HKRDENVI]-C; {77, 77, 6}
(51) Alpha-conotoxin (1) < x(0,35)-{C}(15)-C-C-[SHYNDE]-{C}(2,3)-C-{C}(3,7)-C-{C}(0,12)>; (2)
<{C}(0,14)-C-C-[SHYNDE]-{C}(2,3)-C-{C}(3,7)-C-[G>]> (new); {34, 34, 1}
(52) I-superfamily conotoxin (1) C-{C}(6)-C-{C}(5)-C-C-{C}(1,3)-C-C-{C}(2,4)-C-{C}(3,10)-C (identical);
{37, 37, 0}
(53) Mu-agatoxin and spider toxin SFI (1) C-{C}(2)-[DEKR]-{C}(3)-C-{C}(4,7)-C-C-{C}(2,4)-C-{C}-C-
{C}(4,15)-C-{C}-C-x(0,10)>; {36, 36, 2}
(54) Omega-atracotoxin (ACTX) (1)C-[IT]-P-S-G-Q-P-C (identical); (2)C-C-[GE]-[ML]-T-P-x-C
(identical); {13, 13, 0}
(55) Ergtoxin (1) C-{C}(5)-C-x(8)-C-{C}(2)-C-C-x(9)-C-x(4)-C-{C}-C {25, 25, 0}
Table 1. The conserved peptide patterns similar to PROSITE signatures.

Cytokines and growth factors
(1) Interferon gamma (1) [RHSG]-[KRQ]-A-[AGFYLIVM]-x-[DE]-[LIVFY]-{QPAG}-x-[VI]-[VMLIY]-
{LVIM}-x(1,4)-L-[STAGPKRLIVM]-{Q}-x(1,9)-[AGKR]-[KR]-R; (2) [RHSG]-[KRQ]-A-[AGFYLIVM]-x-
[DE]-[LIVFY]-{QPAG}-x-[VI]-[VMLIY]-{LVIM}-x(1,4)-L-S-P-x(1,7)>; {91, 91, 44}
(2) Interleukin_3 (1) [CVLIM]-[LIVM]-P-x-[AGPST]-x(2)-[STAGDENRKH]-x(12,14)-[DE]-F-[RKQ]-
{NDEAGQST}-K-L; {20, 20, 0}
(3) Interleukin_5 (1) [HDE]-x(2)-C-x(3)-[IVLM]-F-x-G-[LIVMST]-x(2)-L-x-[NST]; {23, 23, 1}
(4) Interleukin_12 alpha (1) [KRHE]-[LM]-C-x(2)-[LM]-[KRHQ]-[AG]-x(3)-R-x(2)-T-x(2)-[KR]-x(3)-Y-
[LMIV]; {34, 34, 7}
(5) Interleukin_15(1)C-{C}(4)-[LM]-{C}-C-[FY]-[LIVFYQ]-x-[DE]-[LIVM]-x(2)-[LIVM]-x(2)-[ED]; {44,
44, 1}


131
(6) Interleukin_17 (1) [RLM]-{QKR}-[PS]-{P}-x-[LIVMFY]-{RKH}-{CP}-[AS]-x-Cx-[CHKRNDESTFY]-
x-[GRKHFY]-C-[LIVM]; {47, 47, 4}
(7) Interleukin_18 (1) [EQ]-[SY]-S-[SL]-x(2)-[GS]-x-[FY]-L-[AST]-[CF]; {41, 41, 3}
(8) Receptivity factor (1) L-[LIVMPAG]-x(2)-[YF]-[LIVM]-x(2)-[QLIVM]-[GA]-x-P-[LIVMFY]-x-
[DENHKRLIVM]-[PAG]-[DEAGST]-[FY]; {204, 204, 0}
(9) GMF-beta (1) [FY]-[LIVM](2)-x-[STAG]-[FYWH]-x(5)-[DE]-x(5)-P-[LIVM]-x(2)-[LIVM]-[FYWN]-
x(2)-P; {29, 29, 1}; {Q9VJL6, Q29NM1}
Hormones
(10) ACTH_domain and opioid neuropeptides (1) K-R-[YF]-G-G-F-[LIVMT]-[STGKRIV]-
[AGKRSTLIVMPY]; (2) K-R-[YF]-G-G-F-[LIVMT]>; (3) K-[KN]-[YF]-G-G-F-M-[KR]; (4) <[YF]-G-G-F-
[LIVMT]-[STGKRIV]-[AGKRSTLIVMPY]; (5){CFYWHM}-Y-x-[MIVSTFY]-{FY}-H-F-R-W; (6) <Y-x-
[MIVSTFY]-{FY}-H-F-R-W; {397, 1045, 4}
(11)FMRFamide and related neuropeptides (1){LCFY}-{LCFYQWST}-{LCFYQWH}-
{LCDEFYKRQW}-[LVMI]-[MLIV]-R-F-G-K-R;(2){LCFY}-{LCFYQWSTLIVM}-{LCFYQWHKR}-
{LCDEFYKRQWLIVM}-[LM]-[MIV]-R-F-GR-[ASPD]-{LCFYHKR}-{LCQST};(3)<x(0,8)-[LVMI]-
[MLIV]-R-F>;(4){CLIVM}-{CAGLIVMW}-{QCFYLW}-[FY]-[MLIV]-R-F-G-K-R; (5){CHIV}-x-{CQN}-
{HIV}-{CLIVMY}-{CAGLIVMW}-{QCFYLWIV}-[FY]-[MLIV]-R-F-G-R-[DNESTAG];(6)<x(0,9)-[FY]-
[MLIV]-R-F>;(7)[AGED]-[LIVMFY]-Q-G-R-F-G-R-[DEN];(8)P-[AGST]-[LIVM]-R-[MLIV]-R-F>;(9)N-
Q-[VI]-R-F-G-K-R; (10) [STG]-[LVMI]-F-R-F-G-K-R; (11)[RD]-[QPH]-F-[FY]-R-F-G-[KR]-{FWYL};
(12)[RD]-[QPH]-F-[FY]-R-F>; (13)R-P-[VI]-G-R-F-G-[KR]-[RS]; (14)S-A-[LM]-A-R-F-G-[KR]-[RS];
(15)[PQ]-[HL]-[LMFY]-R-G-R-F-G-R; (16 )[STNFYH]-[LQ]-PQ-R-F-G-[KR]-{LC}; (17)F-M-[NH]-F-G-
K-R; (18)[AGNQ]-[GLE]-P-[LI]-R-F-G-[KR]-{QLIVMAG}; (19)P-[RK]-P-L-R-F-G>; (20)[FL]-G-T-M-R-
F-G-[KR]-[RS]; (21)Q-[WL]-[LMIV]-[AGKRST]-G-R-F-G-[KR]; (22)[GA]-[GA]-[FY]-[ST]-[FY]-R-F-G-
[RK]; (23)[GA]-[GA]-F-[ST]-[FY]-R-F>; {214,605,2}; {Q7YWT6, Q622X3,Q61P51, Q616K2, Q613X6,
Q21656, P34405, Q60ZQ9, Q618S3, Q620F8, Q620P9, Q7PUD4, Q618T6, Q705J7, Q3SXL4, Q3KNG4,
Q60YH4, Q622X1, Q28Z02, Q297C5, Q28Z02}
(12) Neuropeptide-like protein* (1) G-M-Y-G-G-[FYW]-G-R; (2) A-Q-[FW]-G-Y-G-[GY]-x(2)-
[KRFYG]; (3) G-[FYW]-G-G-Y-G-G-Y-G-R-G; (4) P-L-Q-F-GK-R; (5) [STRIV]-M-S-F-G-K-R; (6)
[AGIV]-M-[AG]-F-G-K-R; (7) [DE]-K-R-G-G-A-R-A-[FYLIVM]; (8) R-x-G-[FML]-R-PG-K-R; (9)
[RFYM]-[AGTR]-F-A-F-A-K-R; {33, 84, 7}; {Q60NA1, Q619H9, Q624T4, Q61BN3, Q627I5, Q60MJ8,
Q625G9, Q622L1, Q622L2}
(13) Wamide neuropeptides* (1) [QRKED]-{P}-[KRPQN]-[IVP]-G-[LM]-W-G-R-[RDESA]; (2)
[ANPRKQ]-x-[AGLQP]-[RHKLIVP]-G-[LM]-W-G-K-R; (3) K-[KR]-x(1,5)-W-x(6)-W-G-[KR]-R; {10, 86,
1} {Q7Q4X3, Q8T3G1, Q60TK2, Q2LZG9}
(14) Thyroliberin (1)[KR]-[HKR]-Q-H-P-G-[KR]-R; {12, 78, 1}
(15) Neurotensin/neuromedin N (1)[KR]-[IVTRK]-P-Y-I-L-K-R; (2) [KR]-[IVTRK]-P-Y-I-L>; {14, 24, 0}
(16)Allatostatin* (1) [KR]-R-{NCKRFY}-x(0,11)-[FY]-[DENAGST]-[FY]-G-[LIVM]-G-[KR]-R; (2)
<x(0,11)-[FY]-[DENAGST]-[FY]-G-[LIVM]>; (3) [KR]-R-x(0,3)-[FY]-[DENAGST]-[FY]-G-[LIVM]>; {52,
222, 3}; {Q7QAG2, Q29BZ8}
(17) Egg-laying hormone (1) K-R-R-[LIVM]-R-F-[HNY]-[KR]-R; (2) P-R-[LIVM]-R-F-[HNY]-
[PSTDEN]-x-[KRG]-[KR]-[KR]; (3) P-R-[LIVM]-R-F-[HNY]-[PSTDEN]-x(1,2)>; {21, 32, 2}
(18)Periviscerokinin (1)<x(0,1)-[AG]-x(0,3)-[GS]-[LIVM]-[LIFY]-x-[FYAMV]-[AGPM]-R-x>;{59, 59, 0}
(19) Somatostatin (1) C-[KRM]-[NSIV]-[FY]-[FY]-W-[KRDE]-[STG]-x-[ST]-x-C; {71, 71, 2}
(20) Orcokinin* (1) [KR]-R-N-F-[DE]-[DE]-[IV]-[DE]-[KR]; (2) <N-F-[DE]-[DE]-[IV]-[DE]-[KR]; {3, 22,
0}; {Q7Q025, Q7QNH4, Q9W1F8, Q292P8}
(21) Allatotropin* (1)N-x(4)-[STIV]-A-R-G-[FY]-G-[KR]-R; (2)N-x(4)-[STIV]-A-R-G-[FY]>; {15, 18, 1};
{Q7QKW9, Q7PZX1}


132
(22) Ghrelin and Motilinrelated peptide (1) G-[STL]-[ST]-F-[LIVM]-[ST]-P-x(0,1)-[AGSTDE]-
[FYQHM]-[QRK]; (2) [FY]-[VILM]-P-x-[FY]-[TS]-x(2)-[DE]-[LIVM]-[QRK]-[RK]-x-[QRK]-[ED]-[KR];
{68, 68, 12}
(23) ADM (1) [AG]-C-{P}-x-[AGFY]-[STMLIV]-C-[AGQIVT]-[VMLIFYHKR]-[QH]-x-[LIVM]; {23, 23,
1}; {Q4RDH7, Q6IFS9}
(24) Hepcidin* (1) C-[CGW]-x-C-C-{C}(4,5)-[CG]-G-x-C-C; {44, 44, 1}; {Q4RUL1, Q4RUL2}
(25) Achatin* (1) K-R-G-F-[AGF]-[DG]-K-R; (2) <G-F-[AGF]-[DG]>; {5, 20, 0}
(26) Cocaine- and amphetamineregulated transcript protein (1) C-x-C-x(5)-C-x(3)-[LIVM]-L-K-[C>];
{11, 11, 2}; {Q4RMR3, Q568S2, Q68EU1, Q4SGG2, Q4T695, Q4TBI3}
(27)Bradykinin (1) P-[PAT]-G-[FW]-[ST]-P-[FL]-R; {58, 84, 7}; {Q5XJ76}
(28) GBP/PSP1/paralytic (1) N-[FY]-x(2)-[GA]-C-x(2)-[GA]-[FY]-x-[RK]-[TS]-x-[DE]-[GA]-[RK]-C-
[KR]-x-[TS]; {18, 18, 0}
(29) Stanniocalcin (1) C-L-x(2,6)-[GA]-C-x(2,5)-F-x-C-x(4)-[ST]-[CS]; {45, 45, 1}
(30) Resistin (1) C-x-C-x(3)-C-x(2)-W-x(7)-C-x-C-x-C-x(4)-W-x(4)-C-C; {22, 22, 2}
(31) Pro-MCH (1) [RK]-R-x(2,6)-[LMIV]-x-C-[MLIV](2)-[GA]-[RK]-[VLIM]-[FY]-x(2)-C-W; (2) R-[ED]-
x(2)-[DE](3)-N-[ST]-[AG]-x-[FY]-[PK]-[IV]-[GD]-[RK]-R; {29, 39, 4}
(32) Pigment dispersing hormone (1) K-R-N-[ST]-[DEGA]-[LIVM](2)-N-[STAG]-[LIVM](2); (2) <N-
[ST]-[DEGA]-[LIVM](2)-N-[STAG]-[LIVM](2); {21, 21, 1}; {Q298P6}
(33) Orexin (1) [HQ]-A-A-G-[IV]-L-T-[LIVM]-G-[KR]-R; (2) [HQ]-A-AG-[IV]-L-T-[LIVM]>; {11, 18, 0}
(34)Leucokinin* (1) [PQAGSTKRH]-x-F-[HYN]-[AGSP]-W-[GA]-G-K-R; (2) <x-[PQAGSTKRH]-x-F-
[HYN]-[AGSP]-W-[GA]>; {11, 11, 0}; {Q60MR3, Q8MNU5}
(35)Myomodulin* (1) [LIVM]-[HQPST]-M-L-R-L-G-K-R; {3, 29, 0}
(36)Nitrophorin (1) C-[ST]-x(9,10)-[KRH]-x(2)-[FYW](2)-x(3,4)-[FYW](2)-x-[TS]-x-[FY]-x(4,5)-[PTS];
{11, 11, 1}
(37)Prokineticin (1) Q-C-x(4)-[CFY]-C-x(2)-[ST]-x(3)-[KR]-x-[LIVM]-[RK]-x-C-x-P-x-[GA]-x(2)-[GA]-
x(2)-C-[HYF]-P; {35, 35, 1}
(38) Leptin (1)L-x-[VIT]-[FY]-[QRH]-[QKA]-[IV]-[LIVMH]-x-[SNG]-[LM]-[PHQS]; {68, 68, 13}
Antimicrobial
(39)Bombinin (1) K-R-[LIVM](2)-G-P-[LIVM](2)-x(2)-[VILM]-[STG]-x(2)-[LIVM]-x(2)-[LIVM](2); (2)
<[LIVM](2)-G-P-[LIVM](2)-x(2)-[VILM]-[STG]-x(2)-[LIVM]-x(2)-[LIVM](2); (3) [SG]-IG-x(0,3)-[LIV]-
x(2,7)-K-[STAGIV]-[AGFYIV]-[LIVF]-[KR]-[GAC]-[AGFYL]-[AGLVIM]-[KRN]; {59, 110, 0}
(40) Brevinin, Dermaseptin, Aurein, Caeridin, Caerin, Dahlein, Temporin Ponericin and Uperin
(1) <x(7)-{C}(2)-x(0,68)-C-[KSTAGLVE]-[LIVA]-[STAKYD]-[KRYGN]-[KRDESTQLG]-C>; (2) C-
[KSTAGLVE]-[LIVA]-[STAKYD]-[KRYGN]-[KRDESTQLG]-C-R-x>; (3) <[DGA]-[LIVF]-[LIVMFW]-
[DNESAGQKPLM]-[STLIVMKFAGDN]-[LVIMAGTF]-[KRAGSTVIL]-[KRHDENGASTQ]-
[LIVMAGKFYSTW]-[IVLMAGFKRH]-[AGKRHSTDENQLIV]-{W}-x(0,2)>; (4) <[DGA]-[LIVF]-
[LIVMFW]-[DNESAGQKPLM]-[STLIVMKFAGDN]-[LVIMAGTF]-[KRAGSTVIL]-
[KRHDENGASTQ]-[LIVMAGKFYSTW]-[IVLMAGFKRH]-[AGKRHSTDENQLIV]-{W}-{CP}(2)-
x(0,35)>; (5) <x(0,45)-{QAGR}-{FYLQKRST}-K-R-[DGA]-[LIVFW]-[LIVMFW]-[DNESAGQKPLFM]-
[STLIVMKFAGDN]-[LVIMAGTFY]-[KRAGSTVIL]-[KRHDENGASTQ]-[LIVMAGKFYSTW]-
[IVLMAGFYKRH]-[AGKRHSTDENQLIV]-{W}-x(0,37)>; (6) <x(0,1)-[FIVLM]-[LIVMFYST]-[PGAQ]-
x-[LIVMFY]-[AGSTIVLM]-[KRSTNDEMLIV]-[LIVMAGFY](0,1)-[LIVMAG](0,1)-x(0,2)-[GKRDEST]-
[LIVM](2)>; (7) K-R-[FIVLM]-[LIVMFYST]-[PGAQ]-x-[LIVMFY]-[AGSTIVLM]-[KRSTNDEMLIV]-
[LIVMAGFY](0,1)-[LIVMAG](0,1)-x(0,2)-[GKRDEST]-[LIVM](2)-G-K>; {278, 310, 25}
(41) Dermorphin (1) K-R-Y-A-F-x-[YVLI]-[PVILM]-x-[RG];(2) <Y-A-F-x-[YVLI]-[PVILM]-x>; {6, 22, 0}
(42)Termicin* (1) C-x(4)-C-W-x(2)-C-x(12)-C-x(4)-C-x-C; {21, 21, 0}


133
(43)Liver-expressed antimicrobial (1) [KR]-P-x(4)-C-x(5)-C-x(3)-[LIVM]-C-[KR]-x(2)-[RKHQ]-[CQ];
{15, 15, 0}; {Q4SXZ9, Q5M9I7}
(44) Penaeidin (1) [CR]-x(1,3)-C-{C}(2)-[LIVM]-{C}(7)-[CYF]-[CST]-{C}(3)-[GA]-x-C-C; {40, 40, 0}
(45) Ceratotoxin* (1) [ST]-[LIVM]-[GA]-[ST]-[AG]-x-[KR]-[KR]-[AG]-[LIVM]-P-[LIVM]-[AG]-[KR](2);
{10, 10, 3}
(46)Attacin (1) [GTS]-[AGVMLI]-[AGFYST](0,1)-[FYLIV]-[AGDEL]-{GMQWKRHNDE}-{PKR}-
[NKG]-[ADENHIV](0,1)-[NDEKR](0,1)-[GSR]-[HFL]-[GAS]-[GAL]-[STAED]-[LIVM]-[TSMQ]-
[KRHDNEGA]-[TSEAG]-[HKRQGT] (2) Y-x-Q-[KRH]-L-[PG]-G-P-Y-G-N-S-x-P; {50, 50, 1}; {Q290V6,
Q291C0, Q295K8, Q29QF8, Q29QG5}
(47)Beta-defensin (1) <x(0,79)-{WP}-x-C-{C}-{CP}-{CW}-{CA}-{C}(0,4)-C-{CP}-{C}-{CW}-{C}(0,2)-C-
{C}(3)-{CP}(2)-{C}(2)-{CP}-{C}(1,5)-C-{C}(0,3)-{C}(4)-C-C-{CDENFWYP}-x(0,128)>; {326, 326, 13};
{Q32P86, Q2XXN6, Q2XXN7, Q2XXN8, Q2XXN9}
(48) 4 kDa defensin* (1) G-[CGA]-P-x(2)-[HQP]-x(2)-[CRK]-[DE]-x-[HP]-[CRWK]-[KR]-G-
[MLIVEDN]; {27, 27, 0}
Toxin
(49)Conotoxin scaffold III/IV, muconotoxin and M conotoxin (1) <x(0,62)-{C}-x(2)-{C}(10)-C-C-
{C}(2,6)-C-{C}(2,5)-C-{C}(1,5)-C-{C}(0,3)-C-{C}(0,3)>; (2) <{C}(0,9)-C-C-{C}(2,6)-C-{C}(2,5)-C-{C}(1,5)-
C-{C}(0,1)-C-{C}(0,3)>; {62, 62, 0}
(50)Conotoxin scaffold IX and tau conotoxin (1) <x(0,49)-{C}(12)-{CDEFY}-{C}(2)-C-C-{C}(4,7)-C-
{C}(0,2)-C-{C}(0,9)>; (2) <{C}(0,14)-C-C-{C}(4,7)-C-{C}(0,2)-C-{C}(0,9)>; {80, 80, 1}
(51)Conotoxin scaffold VI/VII, four-loop conotoxin, Spider potassium channel inhibitory toxin, O
superfamily (1) <x-{PA}-x(0,17)-{C}(0,21)-{C}(2)-{CQ}-{C}(11)-{CI}-{CP}-{C}-{CH}-C-{C}(3,6)-C-{QC}-
{C}(3,9)-C-C-{C}(2,8)-C-{CQ}-{C}(2,9)-C-{C}(0,9)>;(2)<{C}(0,16)-{CQ}-C-{CI}-{C}(2,5)-C-{QPC}-{C}-
{CY}(2)-{C}(0,6)-C-C-{C}(2,8)-C-{CQ}-{C}(2,9)-C-{C}(0,9)>; (3) <C-{CI}-{C}(2,5)-C-{QPC}-{C}-{CY}(2)-
{C}(0,6)-C-C-{C}(2,8)-C-{CQ}-{C}(2,9)-C-{C}(0,9)>; {408, 408, 25}
(52) Scorpion toxin (1) [CKDEN]-{C}(3)-[CI]-{CDEN}-{C}(2)-C-{C}(3)-C-{C}(6,10)-G-{C}(1,2)-[CF]-x-
{C}(3,11)-C-[WYF]-C; (2) [CKDEN]-{C}(3)-[CI]-{CDEN}-{C}(4,9)-C-{C}(3)-C-{C}(6,10)-G-{C}(1,2)-[CF]-
x-{C}(3,11)-C-[WYF]-C; {223, 223, 14}; {Q2TSD9}
(53) Scorpion short toxin 2 (1) C-x-P-C-x(10)-C-x(2)-C-C-x(5,7)-C-x(2,3)-Q-C-LIVM]-C; {14, 14, 0}
(54) Anenome neurotoxin (1) C-x-C-{C}(4)-P-x(6,8)-G-x(5,13)-C-x(6,9)-C-x(6,9)-C-C; {25, 25, 0}
(55) Melittin (1) [LIVM]-[GA]-x(2)-[LIVM]-[KR]-[LIVM]; (2)-x(3)-[LIVM]-P-x-[LIVM](2)-x-W-
[LIVM]; {11, 11, 0}
Table 2. The novel conserved peptide patterns.
Note: each family is described in the following items: (1) the name of the family; (2) all
identified patterns; patterns marked with identical are completely identical to their
PROSITE counterpart and the ones marked as new are novel to PROSITE in Table 1; (3) the
number of true positive peptide or precursor proteins, the number of matches to the pattern,
and the number of false negative hits, all these numbers are in a bracket; (4) if there are
novel putative peptides or precursors predicted by the patterns of the family, they are listed
in a second bracket.
6. Case study
Patterns respectively representing the family of opioid and POMC-derived peptides as well
as the FMRFamide and related neuropeptides (FARPs) are here shown as test cases in order
to provide insights into the conserved sequence characteristics in many know peptide
families and how the peptide patterns deduced based on these characteristics perform.


134
6.1 Opioid and POMC-derived peptides
The family includes subfamilies of opioid peptides and pro-opiomelanocortin (POMC)
proteins, and proteins in this family vary in length ranging from large precursors with a few
hundred amino acids, e.g. Q805B5 in Chimaera phantasma (325aa), to short peptides or partial
sequence fragments, e.g. Q7M2Z6 in Sheep (13aa).
6.1.1 The subfamily of opioid peptides
Opioid peptides are neuropeptides that are involved in pain control mechanisms in
vertebrates, and they consist of proenkephalin (PENK), nociceptin (PNOC) and
prodynorphin (PDYN) (Comb et al., 1982). The 41-column PROSITE pattern PS01252 C-
x(3)-C-x(2)-C-x(2)-[KRH]-x(6,7)-[LIF]-[DNS]-x(3)-C-x-[LIVM]-[EQ]-C-[EQ]-x(8)-W-x(2)-C
matches 39 Uniprot proteins. However, 92 remaining sequences from the subfamily are
disregarded; including nine full peptide precursors e.g. zebrafish Q7T3L0 and 83 peptides or
sequence fragments e.g. human Q9BYY3.
The subfamily is also described by a 71-column Pfam motif PF01160. When querying this
motif against all proteins in the subfamily by means of both global (ls) and fragment (fs)
search modes (http://www.sanger.ac.uk/Software/Pfam/search.shtml), 78 precursors are
singled out. But, the other 53 opioid proteins, e.g. cat Q28409, zebrafish Q8AX66 and
Q9W687 from Acipenser transmontanus, cannot be recognized by the Pfam motif with a score
higher than a gathering threshold.
A further investigation into the proteins missed by the Pfam motif is conducted by
comparing them with all proteins in the non-redundant protein sequence database nr using
BLAST (http://www.ncbi.nlm.nih.gov/BLAST). The alignments with Q28409 (Fig. 2) reveal
that, while the similarities between the two Mammal precursors Q28409 and P01210 are
conserved along the entire sequences, the resemblances between Q28409 and
Q8AX66/Q4RIZ7 from the remote phylum of Actinopterygii are confined to a limited region
identified as [KR]-[KR]-Y-G-G-F-[ML]-[KR]-[KR]. The few highly conserved amino acids
are also observed from the alignments between Q9W687 and Q5Y3C6 from Chondrichthyes
and Q6SYA7 from Dipnoi (Fig. 3). However, this conserved region is too short to produce a
significant score, and therefore BLAST comparison alone will fail to detect the limited
similarity preserved among the distant homologues with a critical confidence level.
The existing PROSITE pattern and the Pfam motif both characterize only the conserved N-
terminal region of the peptide precursors, they are thus not sufficient in identifying all short
bioactive opioid peptides or sequence fragments which are cleaved from their large
precursors and do not carry the N-terminal part of the proteins, but nevertheless bring the
crucial conserved peptide sequence region with them and preserve the fundamental
function of the peptide subfamily. Therefore, although the sequences, e.g. Q28409, Q8AX66
and Q9W687, cannot be identified by the existing motifs, they all share the pattern [KR]-
[KR]-Y-G-G-F-[ML]-[KR]-[KR] from our PeptideMotif database. The pattern, which is
derived from the bioactive peptide sequences, could be more functionally conserved and
more performable in identifying opioid peptides or entire precursor proteins.
6.1.2 The subfamily of POMC-derived peptides
The subfamily shares similar peptide sequences with opioid precursors, but also contains
other non-opioid peptides such as ACTH and alpha-MSH, which are involved in the stress
response and stimulate corticosteroid release (Arends et al., 1998).


135

Fig. 2. Sequence alignments between Q28409 and P01210/Q8AX66/Q4RIZ7 by BLAST.
Notes: the conserved opioid peptide sequence similarities are in bold.
No signature represents the subfamily in PROSITE; three Pfam motifs explain the proteins
including PF08384 (45 columns), PF00976 (41 columns) and PF08035 (31 columns). These
motifs capture separate conserved regions located respectively at the N-ternimus of the
precursors after the removal of the signal peptide, at the sequences coding for ACTH and
for beta-endorphin peptides. However, the remaining parts of the precursors encoding for
peptides of gamma-MSH (12aa) and beta-MSH (17aa) are left untouched. As a result, 27
mature peptides or sequence fragments, e.g. Q9PRN3 from the Sea lamprey, horse P01202
and leech P41989, cannot be detected by any of these Pfam motifs.
Query=Q28409|PENK_FELCA Proenkephalin A-Felis silvestris catus(Mammalia) Length=187

> P01210|PENK_HUMAN Proenkephalin A precursor - Homo sapiens (Mammalia)
Length=267 Score = 429 bits (1004), Expect = 1e-118

Query WETCKEFLKLSQLEIPQDGTSALRESS-PEESHALRKKYGGFMKRYGGFMKKMDELYPQE
WETCKE L+LS+ E+PQDGTS LRE+S PEESH L K+YGGFMKRYGGFMKKMDELYP E
Sbjct WETCKELLQLSKPELPQDGTSTLRENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPME

Query PEEEAP-AEILAKRYGGFMKKDAEEEEDALASSSDLLKELLGPGETETAAAPRGR-----
PEEEA +EILAKRYGGFMKKDAEE+ D+LA+SSDLLKELL G+ R R
Sbjct PEEEANGSEILAKRYGGFMKKDAEED-DSLANSSDLLKELLETGDN------RERSHHQD

Query ---DDEDVSKSHGGFMRALKGSPQLAQEAKMLQKRYGGFMRRVGRPEWWMDYQKRYGGFL
++E+VSK +GGFMR LK SPQL EAK LQKRYGGFMRRVGRPEWWMDYQKRYGGFL
Sbjct GSDNEEEVSKRYGGFMRGLKRSPQLEDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFL

Query KRFADSLPSDEEGESYS
KRFA++LPSDEEGESYS
Sbjct KRFAEALPSDEEGESYS

> Q8AX66|Q8AX66_BRARE Proenkephalin (Fragment) - Brachydanio rerio (Actinopterygii)
Length=216 Score = 140 bits (324), Expect = 9e-32

Query KKYGGFMKRYGGFMKKMDELYPQEPEEEAPAEILAKRYGGFMKKDAE----EEED-----
KKYGGFMKR +E L KRYGGFMKK AE E ED
Sbjct KKYGGFMKR---------------------SESLIKRYGGFMKKAAEFYGLESEDVDQGR

Query ALASSSDLLKELLGP-----GETETAAAPRGRDDED-VSKSHGGFMR-----ALKGSPQL
A+ ++ D+ E+L GE E AA R + E+ +K +GGFMR AL
Sbjct AILTNHDV--EMLANQVEADGEREEAALTRSKGGEEGTAKRYGGFMRRGGLYAL------

Query AQEA--KMLQKRYGGFMRRVGRPEWWMDYQ--KRYGGFLKRFADSLPSDEEGE
E+ + LQKRYGGFMRRVGRP+WW Q KRYGGFLKR S E+ E
Sbjct --ESGVRELQKRYGGFMRRVGRPDWW---QESKRYGGFLKR------SQEQDE

> Q4RIZ7|Q4RIZ7_TETNG Chromosome undetermined SCAF15040 - Tetraodon nigroviridis
(Actinopterygii) Length=246 Score = 123 bits (283), Expect = 2e-26

Query KKYGGFMKRYGGFMKKMD------ELYPQEPEEEA--PAEIL------------------
KKYGGFMKRYGGFM + D E +P +P+EE EIL
Sbjct KKYGGFMKRYGGFMSRRDVPEGALE-HPSDPDEEENIRLEILKILNAAAVHGSEGGGKAG

Query --AKRYGGFMKKDAEEEEDALASSSDLLKELLGPGETETAAAPRGRDDEDVSKSHGGFMR
KRYGGFM++ AEE A+ DLL+ +LG R
Sbjct EEGKRYGGFMRR-AEEG----AAQGDLLEAVLG--------------------------R

Query ALKGSPQLAQEAKMLQKRYGGFMRRVGRPEW--------------WM---DYQKRYGGFL
LK KRYGGFMRRVGRPEW W D QKRYGGF+
Sbjct GLK-------------KRYGGFMRRVGRPEWLVDSSKRGGVLKRAWGSDNDLQKRYGGFM


136

Fig. 3. Sequence alignments between Q9W687 and Q5Y3C6/Q6SYA7 by BLAST.
Note: the conserved opioid peptide sequence similarities are in bold.
The BLAST alignment between Q9PRN3 and all proteins in the nr database unveils that,
although Q9PRN3 cannot be identified by the Pfam motifs, it shares the highly conserved
PeptideMotif pattern Y-x-[MV]-x-H-F-R-W with other POMC subfamily members, e.g.,
Q2L6A9 from Hyperoartia, P01193 and Q53WY7 from Mammalia, and Q32U15 from
Amphibia (Fig. 4). This 8-column peptide pattern is a part of the 41-column Pfam motif
PF00976. While the sequence region, which is described by this Pfam motif, may be an entire
functional or structural domain, this peptide pattern contained within the longer domain is
probably the most essentially functional part.
In total, our procedure identifies six novel peptide patterns in the combination of these two
subfamilies. Among all the 397 proteins in this family, 113 were found to contain two of the
peptide patterns, and the rest match one of them. These patterns characterize conserved
domains located at different regions of a precursor sequence, and each of them can
exclusively represent an opioid or POMC peptide or its precursor protein.
6.2 FMRFamide and related neuropeptides (FARPs)
It is widely known that FARPs occur throughout the whole animal kingdom and therefore
this family is an ideally suited test case to check whether the disclosed pattern is capable of
retrieving FARPs from all metazoan species (Ubuka et al., 2009). In total, 23 conserved
peptide patterns have been uncovered from the family, and they match 214 FARPs
sequences with 605 hits due to the presence of multiple copies of the conserved patterns
within some precursor proteins. The identified FARPs distribute among a wide range of
phyla, including Nematoda (85), Arthropoda (50), Mollusca (24), Annelida (9),
Platyhelminthes (1), Cnidaria (10) and Chordata (35).
An 11-column Pfam motif PF01581 characterizes FARPs from all above-mentioned phyla
except Chordata, e.g. human Q9HCQ7 and mouse Q9WVA8. In addition, conversely to the
PeptideMotif patterns, 49 FARP peptides or precursor proteins in these characterized
phyla, e.g., Q9TWD2 from Lymnaea stagnalis and Q95QP2 from Caenorhabditis elegans, cannot
be revealed by the Pfam motif with a significant score (e-value <0.01).
Query= Q9W687|Q9W687_ACITR Proenkephalin (Fragment)-Acipenser
transmontanus (Actinopterygii) Length=45

> Q5Y3C6|Q5Y3C6_HETPO Proenkephalin - Heterodontus portusjacksoni
(Chondrichthyes) Length=264 Score = 39.2 bits (85), Expect = 0.032

Query 14 RYDGFSKQ------PEHTDSKEITSEEV---EKRYGGFM 43
RY GF K+ P D EI S+EV EKRYGGFM
Sbjct 225 RYGGFMKRWNDILVPSDEDG-EIYSKEVPELEKRYGGFM 262

Score = 31.2 bits (66), Expect = 8.7

Query 14 RYDGFSKQPEHTDSKE--ITSEEVE----------KRYGGFM 43
RY GF K+ DS + I+ EV+ KRYGGFM
Sbjct 105 RYGGFMKK---ADSGDMYIS--EVDDENKGREILSKRYGGFM 141

> Q6SYA7|Q6SYA7_PROAN Prodynorphin (Fragment) - Protopterus annectens
(Dipnoi) Length=191 Score = 33.7 bits (72), Expect = 1.5

Query 33 EEVEKRYGGFM 43
EE++KRYGGFM
Sbjct 169 EELQKRYGGFM 179


137

Fig. 4. Sequence alignments between Q9PRN3 and P01193/Q53WY7/Q32U15 by BLAST.
Note: the conserved peptide sequence similarities are in bold.
The Clustal-W multi-alignment of all these FARP sequences together or within each of the
seven phyla using default parameters (http://www.ebi.ac.uk/clustalw/) shows that the
FARP precursors display sequence similarities within the mature peptide regions,
particularly in the area containing the conserved peptide patterns, and that the remaining
parts of the precursor sequences display rather low similarities. The FARP peptide
precursors also differ from each other by the number of peptide repeat units within the
sequences, which is thought to have arisen by unequal crossover events (Lee et al., 1998). In
addition, we also observed that most of the mature FARP peptides share common C-
terminal sequences but have much mutated N-terminal extensions. All these make it
problematic to construct an accurate multiple alignment in order to derive a statistical

Query= Q9PRN3|Q9PRN3_PETMA Melanotropin MSH-B - Petromyzon marinus
(Hyperoartia) Length=20

> Q2L6A9|Q2L6A9_MORMR Proopiomelanotropin (Fragment) - Mordacia mordax
(Hyperoartia) Length=154 Score = 51.5 bits (114), Expect = 5e-06

Query 2 QESADGYRMQHFRWGQPLP 20
QE+ D YR+QHFRWG+PLP
Sbjct 11 QENPDAYRIQHFRWGEPLP 29

> P01193|COLI_MOUSE Corticotropin-lipotropin precursor(Pro-
opiomelanocortin) (POMC) - Mus musculus (Mammalia) Length=235

Query 8 YRMQHFRWGQP 18
Y M+HFRWG+P
Sbjct 125 YSMEHFRWGKP 135


Query 3 ESADG-YRMQHFRWGQP 18
E DG YR++HFRW P
Sbjct 183 EKDDGPYRVEHFRWSNP 199

Score = 22.3 bits (45), Expect = 2753

Query 8 YRMQHFRW 15
Y M HFRW
Sbjct 77 YVMGHFRW 84

> Q53WY7|Q53WY7_HUMAN Proopiomelanocortin (Fragment) - Homo sapiens
(Mammalia) Length=30 Score = 22.3 bits (45), Expect = 2753

Query 8 YRMQHFRW 15
Y M HFRW
Sbjct 3 YVMGHFRW 10

> Q32U15|Q32U15_9NEOB Proopiomelanocortin A (Fragment) - Trachycephalus
jordani (Amphibia) Length=82 Score = 23.1 bits (47), Expect = 1529

Query 8 YRMQHFRW 15
Y M HFRW
Sbjct 23 YVMSHFRW 30


138
model which represents distantly related proteins from various phyla throughout the
evolutionary history of the FARP peptide family.
7. Conclusion
Protein domains are highly conserved throughout evolution and there are several databases
available that catalogue protein families and domains. Such motif and domain databases are
very useful in assigning a putative function to an unknown protein. Peptide precursor
proteins are a distinctive class of molecules because they undertake various
posttranslational modifications in order to ultimately synthesize stabilized and functional
mature peptides, making the annotation of peptides and peptide precursor proteins
challenging. This is illustrated by the fact that many metazoan peptides and peptide
precursors are not represented by the motifs currently present in the widely used motif
database such as PROSITE.
Because of the tremendously increasing number of protein sequences and because of the
wide range of peptide families, a comprehensive database of conserved patterns typical for
endogenously occurring mature peptides is of great value in identifying new peptides and
precursor proteins to catch up with their sequencing rate. We therefore have designed a
searching procedure to find conserved patterns within the known peptides, and as a result,
we have constructed a PeptideMotif database that is representative of most currently
known peptide families.
Many peptides have been isolated and sequenced as mature peptides and their precursor
proteins are often unknown as yet. Therefore, these small peptides are difficult to be
identified by other motif databases. Motifs in databases such as Pfam contain two Hidden
Marcov Models (HMMs) for each family based on a multiple protein sequence alignment,
one built to find complete domains (ls mode) and the other to match fragments of
domains (fs mode) (Durbin et al., 1998). These motifs are sensitive at identifying complete
domains and thus they can efficiently detect the proteins which have similarities that
cover the full length protein sequence or at least contain a complete domain. However,
these motifs do not work very well when they encounter short peptides which lack
information on amino acids at the sites outside the peptide sequences, or when the
conserved regions are limited, especially in distantly related proteins where the overall-
length sequence similarity may be not well preserved. In contrast, the patterns derived
directly from the mature peptide sequences grasp the highly preserved region of the
precursor proteins and thus are able to identify not only the peptide precursor molecules
but also the fully processed peptides.
Conservative peptide sequence patterns correspond to functionally and structurally
important parts of the peptides, i.e. the binding site to specific receptors, the disulphide
bonds for stability and tertiary structure. The discovery of peptide motifs will be
undoubtedly of great value for any peptide-related studies ranging from the identification
of putative peptides and precursor proteins to the annotation of critical functional
residues (Husson et al., 2010), to the complement of peptidomic research in detecting and
verifying peptides in vitro (Baggerman et al., 2004; Boonen et al., 2008; Menschaert et al.,
2010). For example, scanning the peptide patterns against Uniprot revealed 95 proteins
(listed in Tables 1 and 2) which are not as yet annotated as putative peptides or precursor
proteins.


139
When determining short functional patterns for peptide sequences, we have to evaluate how
representative the peptide motifs are in the 110 characterized peptide families. Short motifs
often have some degree of degeneracy and the presence of a motif in a protein may reflect a
conserved functional role, a yet to be discovered structural functional role or a non-
functional role. When using the short currently identified peptide patterns, while the false
positives are kept to zero, we observe that 440 (3.8%) of the mature peptides or sequence
fragments and 282 (2.5%) of the peptide precursor proteins in these described families
cannot be recognized by the peptide patterns. Many of them could be determined by
combining the peptide pattern search procedure with the structural hallmarks of bioactive
peptides and their precursors (Liu et al., 2006), such as the length of a peptide precursor
which is usually not longer than 500 amino acids, the presence of a signal peptide which
directs a precursor protein into the secretary pathway of the cell, and the presence of typical
cleavage sites flanking the mature peptides. To be even more successful in identifying all
false negatives while eliminating all false positives because of the short length and
degeneracy of most short motifs, it may be possible to make use of 3D structural patterns
when they become available for peptide precursor proteins. Patterns that integrate 3D
structural information of the sequences will be more sensitive in identifying peptides and
peptide precursors (Gribskov et al., 1988; Taylor et al., 2004).
While the majority of known peptide families have been profiled by the established peptide
patterns, the remaining ones accounting for in total 251 peptides and precursor proteins (2%
of all the proteins in the peptide-precursor database) are not processed by the pattern search
procedure. They are from small peptide families, such as eclosion hormones, ecdysis-
triggering hormones and apelin, which have only a few homologies so far. A pattern based
on the small number of peptides usually cannot gain enough confidence in representing the
family, and also cannot sufficiently reflect the sequence divergence accumulated in the
evolutionary course of the family member. As more peptides and precursor proteins are
sequenced, our patterns search procedure can be applied to the corresponding families and
the PeptideMotif database will be updated accordingly, keeping the peptide pattern
database widely applicable for the identification of critical functional residues and for the
annotation of hypothetical molecules in various peptide families.
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7
Database Mining: Defining the Pathogenesis of
Inflammatory and Immunological Diseases
Fan Yang, Irene Hwa Yang, Hong Wang and Xiao-Feng Yang
Department of Pharmacology, Cardiovascular Research Center, Temple University
School of Medicine, Philadelphia,
U.S.A.
1. Introduction
Cardiovascular disease (CVD) is a leading cause of mortality in developed countries (Jan et
al., 2010; Yang et al., 2008). Despite a long held understanding and strong characterization of
the traditional and non-traditional risk factors for CVD, some mechanisms of CVD onset
have only recently been uncovered. As a chronic inflammatory autoimmune disease,
atherosclerosis and its progression involve innate and adaptive immune systems. Using new
concepts and technologies to improve the current understandings of the molecular
pathogenesis of inflammatory and immune responses would lead to the future development
of novel therapeutics for these diseases.
Biomedical literature and databases, available in electronic forms, contain a vast amount of
knowledge resulting from experimental research (Ishii et al., 2007; Palakal et al., 2007). In the
past decade, both traditional hypothesis-driven research and discovery-driven -omics
research, including genomics, transcriptomics (Liang et al., 2005), proteinomics,
metabolomics, glycomics, lipidomics, localizomics, protein-DNA interactomics, protein-
protein interactomics, fluxomics, phenomics (Joyce & Palsson, 2006), and antigen-omics
(http://www.cancerimmunity.org/links/databases.htm) (Houle et al., 2010; Shimokawa et
al., 2010; Weinstein, 1998;2002), has generated a tremendous amount of data and established
many experimental data-based searchable databases. These databases include PubMed,
nucleotide database, protein database, and other databases generated by the National
Institutes of Health (NIH)/National Center for Biotechnology Information (NCBI) (see the
NCBI handbook at http://www.ncbi.nlm.nih.gov/books/NBK21101/) and other
institutions. This development has not only provided resources, but also raised
unprecedented challenges and opportunities for biomedical scientists to develop more
systemic and panoramic approaches to analyze the data contained in the databases and
generate new hypotheses. The inconsistency between the vast amount of experimental data,
various searchable databases, and relatively smaller numbers of database-mining research
papers (< 50 papers on database mining in inflammation and immune responses listed in
the PubMed) indicate the challenges that experimental biomedical scientists face, which
include both technical/methodological difficulties and out-of-date concepts.
Traditionally, medical literature search using the Index Medicus was the major approach for
biomedical scientists to identify knowledge gaps and preparing new hypotheses. However,
this approach has been significantly enhanced by more systemic approaches such as 1)


144
NCBI-PubMed search and Google Scholar search; 2) experimentally screening cDNA
libraries and various arrays (nucleic acid arrays, antibody arrays, protein arrays and
metabolic arrays) (King et al., 2005; Loza et al., 2007; Pandey et al., 2004; Warner &
Dieckgraefe, 2002); and 3) mining experimental databases (Chen et al., 2010; Jan et al., 2010;
Ng et al., 2004; Yang et al., 2006a; Yang et al., 2006b; Yin et al., 2009). The screening analysis
of microarray data often requires bioinformatic methods, algorithms, and expertise. In
comparison, database mining offers many advantages. First, database mining requires much
less bioinformatic assistance in each laboratory when compared to the generation of
algorithms required in microarray analyses, since the purpose of generating databases is to
use bioinformatic approaches to mine easily organize the experimental data for biomedical
scientists to mine (Spasic et al., 2005). Second, database mining enables full-value extraction
from costly experimental data, and third, it provides panoramic analyses on existing
knowledge gaps by generating new hypotheses for further experimental research. However,
database mining requires biomedical scientists to have more conceptual advances than
technical assistances. The purpose of database mining is to analyze experimental data
deposited by various research projects, rather than predicting theoretic results based on
pure theoretical bioinformatic studies. Thus, database mining is not limited to sequence
comparisons of nucleic acids and proteins (Mount, 2004), sequence alignments, analysis of
hydrophobicity index and functional domain prediction of proteins. Additionally, database
mining has not generally been listed as a required course for graduate and postdoctoral
studies, which presents a challenge of properly training young biomedical scientists with
essential database mining techniques. On top of these aforementioned challenges, reviewers
from peer-reviewed database mining publications often mistakenly regard the experimental
data in electronic forms deposited in databases as non-experimental or theoretical and
demand ridiculous additional verifying experiments to be performed, even requiring the
use of outdated experimental techniques or methods. To overcome these difficulties,
bioinformatic scientists will have to work together with biomedical colleagues and delve
into the biological significance of database mining projects, rather than sticking to an
argument of no algorithms means no bioinformatics. Already, more and more database
mining papers have been published as scientists put aside their differences. For example, the
2011 (18
th
) database issue of the journal Nucleic Acid Research features descriptions of 96
new and 83 updated online databases covering various areas of molecular biology (Galperin
& Cochrane, 2011). The Nucleic Acids Research online Database Collection, available at:
http://www.oxfordjournals.org/nar/database/a/, now lists 1330 carefully selected
molecular biology databases. In addition, 32 databases and analysis resources of
immunological interest have been established (Salimi et al., 2010). Moreover, our recent
invited review lists 11 B cell antigen epitope databases and 13 T cell antigen epitope analysis
resources (Jan et al., 2010). These progresses suggest that a data mining approach has
gradually been accepted as mainstream practice in analyzing experimental data and
generating new hypotheses for various projects (Salimi et al., 2010).
Our lab has successfully pioneered major advances in database mining in the fields of
adaptive immune reactions, innate immune responses, and inflammation (Chen et al., 2010;
Jan et al., 2010; Ng et al., 2004; Virtue, 2011; Yang et al., 2006a; Yang et al., 2006b; Yin et al.,
2009). In this chapter, we will summarize the general approaches, principles, and databases
used and new working models proposed in our database mining research. This discussion
will prove to be important and useful for most biomedical scientists, since many are not

Database Mining: Defining the Pathogenesis of Inflammatory and Immunological Diseases

145
often involved in the bioinformatic algorithm generation, but may want to use database
mining methods in their research either as parts of existing experimental studies or as free-
standing projects. Of note, the database mining concept is not brand new. Medical
research has a long history in full-value extraction from costly data. For example, a meta-
analysis uses a statistical approach to combine the results of several epidemiological studies
that address a set of related research hypotheses. This practice started well over 100 years
ago and has been widely used in various disease-related researches
(http://en.wikipedia.org/wiki/Meta-analysis) (Egger & Smith, 1997; Egger et al., 1997). We
believe that the practice of database mining will become a routine exercise to identify
existing knowledge gaps and to generate new hypotheses.
2. Principles of database mining
In recent years, many databases regarding immune responses and inflammation have been
established (Jan et al., 2010; Yang et al., 2006a), which have expanded the scope and depth of
a publicly searchable online repertoire of tools. The results derived from the database
mining analyses have become parts of many research papers or free-standing papers.
Although projects may vary in format, database mining approaches follow the same set of
principles (Fig. 1): 1) Hypothesis: A clearly-presented hypothesis based on the current
biomedical literature search in a given field and previous experimental data in the lab is
required to carry on a database mining project as we reported (Ng et al., 2004; Yan et al.,
2004), which is similar to that of experimental projects. Of note, the database mining referred
here focuses on database mining as a free standing project rather than as a part of
experimental research; 2) Scope: Database mining scopes in terms of gene numbers are far
more than that examined in experimental approaches. For example, our own research will
examine mRNA transcript expressions of about 30 genes including all the reported toll-like
receptors, NOD-like receptors, and inflammatory caspases in more than ten tissues. This
scope allows us to obtain a panoramic view on the expressions of inflammatory pathways
without focusing on a single gene in many tissues (Yin et al., 2009); 3) Suitable databases:
Databases that are suitable for examining the hypothesis are available for online analytic
search, which is also similar to the methods and reagents for experimental projects; 4)
Sizable experimentally verified data for generating confidence intervals with statistical
significance: To consolidate the results generated from database mining, the experimentally
verified data are published by various laboratories, which can be used to generate
statistically significant confidence intervals by using the same online analysis tools as we
reported (Virtue, 2011). In this study, our analysis in the TargetScan yielded 524
microRNAs, which were predicted to participate in 1368 unique interactions with the 33
inflammatory gene mRNAs. To ensure relevance, we examined the context value and
percentage of experimentally verified microRNAs. Confidence intervals were generated
from 45 interactions between 28 experimentally verified human microRNAs and 36 genes
found within the Tarbase, an online database of experimentally verified microRNAs
(http://diana.cslab.ece.ntua.gr/tarbase/) (Papadopoulos et al., 2009; Sethupathy et al.,
2006). These experimental interactions were also selected based on their confirmation by
luciferase reporter assays and single site specificity. The 45 microRNA-mRNA interactions
that met these criteria were then evaluated in TargetScan to determine the microRNA


146
Develop a well-presented hypothesis based on experimental data and
analysis on current knowledge gap
Determine the scope, focus, and pathway for database mining
Find suitable databases that contain experimental data, which are
organized with appropriate bioinformaticexpertise
Find sizable experimental data for generating confidence intervals,
which are consolidated with statistical tools of sample size
determination
Identify verifiable experimental methods
Propose a new working hypothesis/working model for further
experimental research

Fig. 1. Detabase mining flow-chart and principles.
context values and percentages. Analysis of this data yielded a mean and standard deviation
(SD) of -0.25 0.12 and 76.07 19.07 for context value and context percentage, respectively.
The intervals were then constructed and the lower limits (the mean - 2 x standard
deviations) were calculated for context percentage (76.07-1.96 (19.07/SQRT (46)) = 76.07 -
5.51 = 70.56) and context value (-0.25-1.96(0.12/SQRT (46) = - 0.25 - 0.04= -0.22). All
predicted microRNAs interactions with a context value -0.22 and context percentage 70
were accepted. Using the lower limit thresholds for context value and percentage, 297 out of
the 524 predicted microRNAs met the criteria and were considered equivalent to the
experimentally verified microRNAs. In order to generate valid confidence intervals, sample
sizes have to be estimated with statistical tools of sample size determination (Rosner, 2000)
as we reported (Ng et al., 2004); 5) Verifiable methods: Experimental methods are available


147
to verify the data generated by the database mining (Yan et al., 2004); and 6) A new working
model/hypothesis: Through database mining, a new knowledge gap will be identified, and
a new hypothesis will be proposed to test fewer, much more-focused genes in further
experiments. The following sections will illustrate these principles in our own publications
(Chen et al., 2010; Jan et al., 2010; Ng et al., 2004; Virtue, 2011; Yang et al., 2006a; Yang et al.,
2006b; Yin et al., 2009).
3. Database mining example 1: Stimulation-responsive alternative splicing is
an important mechanism in generating self-antigen epitopes (Ng et al., 2004;
Xiong et al., 2006; Yan et al., 2004; Yang et al., 2006a; Yang, 2007)
In our invited review, we pointed out that the identification and molecular characterization of
self-antigens expressed by human malignancies, that are capable of elicitation of anti-tumor
immune responses in patients, have been an active field in tumor immunology (Yang & Yang,
2005). More than 2,000 tumor antigens have been identified, and most of these antigens are
self-antigens (Yang & Yang, 2005). Despite this, the important question of how non-mutated
self-protein antigens, generated from normal cells and tumor cells, gain immunogenicity and
trigger immune recognition remained unanswered (Yang & Yang, 2005). Mutations may be
responsible for some aspects of elevated immunogenicity underlying certain tumor-specific
antigens (p53 and Ras), while chromosome translocations and abnormalities, such as
expression of the fusion oncogene Bcr-Abl in chronic myelogenous leukemia (Clark et al., 2001;
Pinilla-Ibarz et al., 2000; Yotnda et al., 1998; Zorn, 2001) (Yang et al., 2002; Yang et al., 2001) are
responsible for other aspects. However, the mechanism underlying the immunogenicity of
most non-mutated self-tumor antigens is their aberrant overexpression in tumors (Yang &
Yang, 2005). Zinkernagel et al (Zinkernagel & Hengartner, 2001) suggested that the
overexpression of self-antigens or novel antigenic structure, overcomes the threshold of
antigen concentration at which an immune response is initiated (Shlomchik et al., 2001). This
threshold might be lower for certain untolerized regions of certain antigen epitopes.
Overexpressed genes, often encode tumor antigens up to 100 fold. These genes are identified
by serological identification of self-antigens by screening a cDNA library with patients sera
(SEREX) (Sahin et al., 1995), which may reflect the inherent methodological bias for the
detection of abundant transcript (Preuss et al., 2002). The overexpression of tumor antigens in
tumors may result from transcriptional and post-transcriptional mechanisms. We recently
demonstrated that overexpression of tumor antigen CML66L in leukemia cells and tumor cells
via alternative splicing is the mechanism for its immunogenicity in patients with tumors (Yan
et al., 2004; Yang et al., 2001). This not only illustrates the principle of overexpression of tumor
antigen, but also elucidated alternative splicing as its molecular mechanism (Yan et al., 2004).
A significant proportion of the SEREX-defined self-tumor antigens are autoantigens (Chen,
2004), for example, CML28 that we identified is autoantigen Rrp46p (Yang et al., 2002). Using
this information gathered from SEREX, we hypothesized that alternative splicing is a general
mechanism for the overexpression of untolerized self-antigen epitopes in tumors and
autoimmune diseases. In order to test this hypothesis, we database mined the NIH-NCBI
AceView database to examine the potential mechanisms of how non-mutated self-proteins
gain new untolerized structures that trigger immune recognition (Ng et al., 2004). The
AceView database provides a curated, comprehensive, and non-redundant sequence
representation of all public mRNA sequences (mRNAs from GenBank or RefSeq, and single
pass cDNA sequences from dbEST and Trace). These experimental cDNA sequences are first


148
co-aligned on the genome, and then clustered into a minimal number of alternative transcript
variants and grouped into genes (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/).
Our results showed that alternative splicing occurs in 100% of autoantigen transcripts. This is
significantly higher than the approximately 42% rate of alternative splicing observed in the
9554 randomly selected human gene transcripts (p<0.001). Within the isoform-specific regions
of the autoantigens, 92% and 88% encoded MHC class I and class II-restricted T-cell antigen
epitopes, respectively, and 70% encoded antibody binding domains. Alternative splicing can
be canonical or non-canonical. Canonical splicing removes introns that have 5GT and 3AG
consensus flanking sequences (GT-AG rule) (Lewin, 2000). Our results demonstrated that 80%
of the autoantigen transcripts undergo non-canonical alternative splicing, which is
significantly higher than the less than 1% rate in randomly selected gene transcripts (p<0.001).
These studies suggest that non-canonical alternative splicing may be an important mechanism
for the generation of untolerized epitopes that may lead to autoimmunity. Furthermore, the
product of a transcript that does not undergo alternative splicing is unlikely to be a target
antigen in autoimmunity (Ng et al., 2004). To consolidate this finding, we also examined the
effect of proinflammatory cytokine tumor necrosis factor- (TNF-) on the prototypic
alternative splicing factor (ASF)/SF2 in the splicing machinery. Our results show that TNF-
downregulates ASF/SF2 expression in cultured muscle cells. This result correlates with our
finding of reduced expression of ASF/SF2 in inflamed muscle cells from patients
with autoimmune myositis (Xiong et al., 2006). Based on our and others data, we
recently proposed a new model of stimulation-responsive splicing for the selection of
autoantigens and self-tumor antigens (Yang et al., 2006a) [also see Fig. 1 at
(http://preview.ncbi.nlm.nih.gov/pubmed/16890493)]. Our new model theorizes that the
significantly higher rates of alternative splicing of autoantigen and self-tumor antigen
transcripts that occur in response to stimuli, such as proinflammatory cytokines, could induce
extra-thymic expression of untolerized antigen epitopes to elicit autoimmune and anti-tumor
responses. By using B lymphocyte (B cell) antigen epitope analysis databases and T cell
antigen epitope analysis databases listed in Tables in our recent invited review
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858284/pdf/JBB2010-459798.pdf) (Jan et
al., 2010), we showed that protein sequences encoded by alternatively spliced exons are
sufficient to equip antibody-binding antigen epitopes and major histocompatibility complex
(MHC) class I- and MHC II-restricted T cell antigen epitopes to stimulate B lymphocytes and T
lymphocytes, respectively (Ng et al., 2004). Of note, our model not only applies to non-
mutated self-tumor antigens associated tumors and autoantigens associated with various
autoimmune diseases, but also to the composition and expansion of the self-antigen repertoire
of stem cells. Our additional database mining study has generated a new model of differential
epitope processing for MHC class I-restricted viral antigen epitopes and tumor antigen
epitopes (Yang et al., 2006b). Our reports have demonstrated the principles of database mining
in adaptive immune responses.
4. Database mining example 2: Three-tier model for inflammasome/caspase-1
activation and inflammation privilege of tissues are important mechanisms
underlying the differences in the readiness of inflammation initiation in
tissues
Atherosclerosis is the leading cause of morbidity and mortality in industrialized society.
Several traditional risk factors have been identified for atherosclerosis including


149
hyperlipidemia, oxidized low density lipoprotein, cigarette smoking, diabetes,
hypertension, obesity (Ross, 1992), and hyperhomocysteinemia (HHcy), etc. Chronic
vascular inflammation is an essential requirement for the progression of atherosclerosis in
patients (Hansson, 2005). Recent progress in characterizing pathogen-associated molecular
patterns (PAMPs) receptor families (PAMP-Rs) and inflammasomes (the protein complex
for activation of caspase-1) has further emphasized the importance of proinflammatory
cytokine interleukin-1 (IL-1) signaling in bridging proatherogenic risk factors to initiate
inflammation (Yang et al., 2008). However, constitutive expression levels and expression
readiness of PAMP-Rs, inflammasome components and proinflammtory caspases in tissues
remained poorly defined. We hypothesized that PAMP-Rs, inflammasome components,
proinflammatory caspases, IL-1, and IL-18 are differentially expressed in cardiovascular
tissues. To examine this hypothesis, we mined the NCBI-UniGene database, analyzed cDNA
cloning and DNA sequencing data from tissue cDNA libraries and studied expression
profiles of Toll-like receptors (TLRs), cytosolic nucleotide binding and oligomerization
domain (NOD)-like receptors (NLRs), inflammasome components, inflammatory caspases,
and caspase-1 cleavable inflammatory cytokines. The UniGene database provides an
organized view of the transcriptome with information on protein similarities, gene
expression, cDNA clone reagents, and genomic location
(http://www.ncbi.nlm.nih.gov/unigene), in which each UniGene entry is a set of transcript
sequences that appear to come from the same transcription locus (gene or expressed
pseudogene). After analyzing the data from the UniGene database, we made several
important findings: (1) Among 11 tissues examined, vascular tissues and heart express fewer
types of TLRs and NLRs than immune system tissues including blood, lymph nodes,
thymus, and trachea; (2) Brain, lymph nodes, and thymus do not express proinflammatory
cytokines IL-1 and IL-18 constitutively, suggesting that these two cytokines need to be
upregulated in response to inflammatory stimuli in the tissues; and (3) based on the
expression data of three characterized inflammasomes (NALP1, NALP3 and IPAF
inflammasomes), the examined tissues can be classified into three tiers: the first tier tissues
including brain, placenta, blood, and thymus express inflammasome(s) in constitutive
status; the second tier tissues have inflammasome(s) in nearly-ready expression status (with
the requirement of upregulation of one component); and the third tier tissues like heart and
bone marrow, require upregulation of at least two components in order to assemble
functional inflammasomes. Based on the expression readiness of inflammasomes in tissues,
we propose a new working model of three-tier responsive expression of inflammasomes in
tissues and suggest a new concept of third tier tissues inflammatory privilege, which
provides an insight on the differences of tissues in initiating acute inflammations. This
model suggests that (a) first-tier tissues with constitutively expressed inflammasomes
initiate inflammation quicker than second and third-tier tissues; and (b) second tier tissues
(requiring one component of upregulation) including vascular tissue, and third tier tissues
including heart (requiring more than one component upregulation) are in an inducible
expression status of inflammasomes. The inducible expressions of inflammasomes are
presumably mediated through various signal pathways that initiate inflammation, and the
interplay between the signal pathways, may take a longer time and overcome a higher
threshold than first tier tissues. Traditional concepts of immune privilege suggests a
protective mechanism from autoimmune destruction based on the lack of expression of
antigen-presenting self-major compatibility complex (MHC) molecules in tissues (Yang &
Yang, 2005). The lack of expression of self-MHCs in immune privileged tissues including


150
testis results in the failure of self-antigen presentation that stimulates the hosts immune
system, thereby protecting immune privileged tissues from autoimmune destruction.
Similarly, we proposed a new concept of tissues inflammatory privileges that emphasize a
protective mechanism against tissue destruction mediated by inflammasome/IL-1-based
innate immune responses. In our new concept of tissues inflammatory privilege, vascular
tissue and heart disproportionally express fewer types of TLRs and NLRs and may only
inducibly express inflammasomes, thus preventing against uncontrolled inflammatory
destruction mediated by inflammasome-based innate immune responses (Streilein & Stein-
Streilein, 2000). Our new concept and model may also explain the potential differences
between cardiovascular tissues and other tissues in initiating acute inflammation. The first-
tier tissues may have a higher probability of experiencing acute inflammation than the
second-tier and third-tier tissues.
We and others showed that elevated levels of plasma homocysteine (Hcy), termed
hyperhomocysteinemia (HHcy), is an independent risk factor, equivalent to hyperlipidemia,
for cardiovascular diseases (CVD) including coronary heart disease and stroke (Maron &
Loscalzo, 2009; Wang et al., 2003; Zhang et al., 2009). Recently, we performed an additional
database mining study using to examine the expression of more than 20 homocysteine
metabolic enzymes and methylation enzymes in >20 tissues in humans and mouse (Chen et
al., 2010). We generated a new model of how hypomethylation (a post-translational protein
modification) modulates the expressions of homocysteine-metabolizing enzymes (Chen et
al., 2010). Taken together, our studies have demonstrated the principles of database mining
in innate immune reactions.
5. Database mining example 3: A group of anti-inflammatory microRNAs may
play critical roles in inhibiting the expression of proatherogenic molecules
Previous research has established that numerous genes are upregulated in atherogenesis
through epigenetic or genetic transcriptional mechanisms (Turunen et al., 2009). However,
transcription-independent mechanisms have received far less scrutiny. Recent publications
suggest that microRNAs, a newly characterized class of short (18-24 nucleotide long),
endogenous, non-coding RNAs (Bartel, 2009), contribute to the development of particular
disease states by regulating diverse biological processes such as cell growth, differentiation,
proliferation, and apoptosis (Zhang, 2008). This biological control is accomplished by post-
transcriptional gene silencing (Naeem et al., 2010) through Watson and Crick base-pairing
predominately at the 3-untranslated region (3UTR) of messenger RNAs (mRNAs) (Cordes
et al., 2009; Rasmussen et al., 2010). This pairing can be further characterized as perfect or
near perfect, leading to target mRNA cleavage and degradation, or imperfect, causing
the inhibition of mRNA translation (Naeem et al., 2010). With the identification and
sequencing of more than 800 human microRNAs thus far, it is thought that up to 30% of
human genes may be regulated by microRNAs (Cheng et al., 2010; Zhang, 2008). Supporting
evidence suggests that microRNAs function as key players during critical stages of cellular
development and finely tune gene expression in the maintenance of routine cellular
functioning (Baek et al., 2008). Furthermore, microRNAs can act on transcription factors,
which lead to a broad indirect cellular effect as a result of their widespread gene modulating
nature. In addition, the recent research has demonstrated that changes in microRNAs
expression patterns are connected to several pathological conditions including
cardiovascular disease and atherosclerosis. These studies primarily focused on


151
characterizing microRNAs in atherosclerosis disease models, which had been previously
reported to have elevated expression in disease conditions (Haver et al., 2010; Rink &
Khanna, 2010). Thus, current microRNAs research has failed to provide a panoramic view of
how microRNAs regulate proatherogenic inflammatory genes in a panoramic view and
whether upregulation of proatherogenic inflammatory genes is the result of anti-
inflammatory microRNA downregulation. To address these issues, we hypothesized that a
group of anti-inflammatory microRNAs may regulate the expressions of proatherogenic
molecules (Virtue, 2011). We then developed a novel database mining approach using three
types of databases including the online microRNA target prediction software TargetScan
(http://www.targetscan.org/) (Dong et al., 2010; Rosero et al., 2010; Vickers & Remaley,
2010), the Tarbase, an online database of experimentally verified microRNAs
(http://diana.cslab.ece.ntua.gr/tarbase/) (Papadopoulos et al., 2009; Sethupathy et al.,
2006), and the online microRNA.org expression database (http://www.microrna.org/
microrna/home.do) (Betel et al., 2008), in concert with a statistical analysis strategy
established in our previous database mining publications (Chen et al., 2010; Ng et al., 2004;
Shen et al., 2010; Yang et al., 2006b; Yin et al., 2009). Our unique research using database
mining yielded several key findings. First, we discovered that the expression of 33
inflammatory genes (mRNAs) is upregulated in atherosclerotic lesions and second, that the
mRNAs of those genes contain structural features in their 3UTR for potential regulation by
microRNAs. Furthermore, these structural features are statistically identical to
experimentally verified 3UTR microRNAs binding sites. Third, 21 out of the 33
inflammatory genes (64%) are targeted by highly expressed microRNAs while the
remaining 12 inflammatory genes (36%) are targeted by normally expressed microRNAs.
Fourth, it was also established that 10 of the 21 highly expressed microRNA-targeted
inflammatory genes (48%) were targeted by a single microRNA, suggesting the specificity of
microRNA regulation. Meanwhile, 12 out of the 25 highly expressed microRNAs (48%)
targeted single inflammatory genes while the other 13 microRNAs targeted multiple
inflammatory genes. Finally, it was determined that the microRNAs targeting
atherosclerotic inflammatory genes use statistically higher numbers of poorly conserved
binding interactions than the control group of microRNAs from the confidence interval.
These results suggest that the microRNAs regulating atherosclerotic inflammatory genes
possess special features (Virtue, 2011).
Previous research has shown that microRNAs participate in modulating atherosclerosis-
related processes including hyperlipidemia (microRNA-33, microRNA-125a-5p),
hypertension (microRNA-155), plaque rupture (microRNA-222, microRNA-210), and
atherosclerosis itself (microRNA-21, microRNA-126) (Rink & Khanna, 2010). However,
whether certain microRNAs play a role in preventing the disease development remains
unknown. One of the most interesting findings from our study is that the 25 microRNAs
that are highly expressed under normal untreated conditions target 21 out of the 33 (64%)
atherosclerosis-upregulated inflammatory genes. The important result suggests a novel
mechanism where a group of highly expressed anti-inflammatory microRNAs suppress the
upregulation of proatherogenic inflammatory genes under normal physiological conditions.
It has been well established that microRNAs play important roles in fine-tuning
developmental processes and participate in the development of diseases such as cancer. Our
results are the first to suggest that microRNAs may play a protective role by suppressing
proatherogenic genes to maintain healthy arteries. Our conclusion is supported by other
publications, which show that 7 out of the 20 microRNAs identified in this study were


152
downregulated in the experimental studies by various proatherogenic factors (Chen et al.,
2009; Elia et al., 2009; Ji et al., 2007). Together, our studies have demonstrated the principles
of database mining in inflammation.
6. Conclusion
Active research in human and mouse genomes, transcriptomes, microRNAs transcriptomes,
proteomes, and antigen-omes in the past decade has generated a tremendous amount of
data and established many experimental data-based searchable databases. This provides
unprecedented opportunities for biomedical scientists to develop more systemic and
panoramic approaches to analyze the databases and generate new hypotheses. In this
chapter, we briefly summarize our pioneering efforts in using our new database mining
methods to address important questions in inflammatory and immunological diseases. The
new principles and basic methodologies of database mining developed in our laboratories
are elucidated in the following studies: 1) stimulation-responsive alternative splicing model
for the generation of untolerized autoantigen epitopes; 2) a three-tier model for
inflammasome/caspase-1 activation and inflammatory privileges of tissues; and 3) a group
of anti-inflammatory microRNAs in inhibiting proatherogenic gene expression during
atherogenesis. With recent technological breakthroughs, database mining has provided
significant new insights and hypotheses in specifying the novel directions for experimental
research.
7. Acknowledgements
This work was partially supported by the National Institutes of Health Grants HL094451
and HL108910 (XFY), HL67033, HL82774, and HL77288 (HW). FY and IHY contribute
equally to this work. Correspondence: Prof. Yang at xfyang@temple.edu.
Disclosures: none declared.
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8
Data Mining Pubmed Identifies Core Signalings
and miRNA Regulatory Module in Glioma
Chunsheng Kang
1
et al.

*
1
Department of Neurosurgery, Laboratory of Neuro-Oncology, Tianjin Medical University
General Hospital, Tianjin Key Laboratory of Nerve Injury, Variation and Regeneration,
Tianjin,
China
1. Introduction
Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer and
persist as serious clinical and scientific problems. The current standard of therapy for GBM
patients, include surgery, radiotherapy and chemotherapy with temozolomide, produces a
median survival of only 14.6 months (Stupp et al., 2005). Now, new intervention is
increasingly being tested, particularly with inhibitors of neo-angiogenesis and growth factor
receptors, and high throughout profiling studies are leading to the discovery of novel
genetic alterations and signaling pathways. The Cancer Genome Atlas Network recently
catalogs recurrent genomic abnormalities in GBM, and proposes a molecular classification of
GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrates
multidimensional genomic data to establish patterns of somatic mutations and DNA copy
number (Verhaak et al., 2010). In recent years, microRNAs (miRNAs), small noncoding RNA
molecules, have been identified in the progression of various human cancers and used to a
notable molecular label to cancers. In glioma, miR-21, miR-221, miR-222, miR-181a and miR-
125b have been proven to play critical roles in gliomagenesis and proposed as novel targets
for antiglioma therapies (Shi et al., 2008; Shi et al., 2010; Zhang et al., 2009b; Zhang et al.,
2010c; Zhou et al., 2010a; Zhou et al., 2010b). Thus, molecular regulation of glioma is
comprehensive and still unclear and under further investigation.
Biomedical literature is growing at a double-exponential pace, with approximately 20
million publications in MEDLINE. Up to now, there have been more than 50 thousand of
glioma-related publications in MEDLINE (Pubmed with: glioma). Thus, a massive wealth of
information is embedded in the literature and waiting to be discovered and extracted.
Literature mining is a promising strategy to utilize this untapped information for
knowledge discovery and has been applied successfully to various biological problems
including the discovery and characterization of molecular interactions (protein-protein,
gene-protein, gene-drug, protein sorting and molecular binding) (Friedman et al., 2001;

*
Junxia Zhang
1
, Yingyi Wang
2
, Ning Liu
2
, Jilong Liu
3
, Huazong Zeng
3
, Tao Jiang
4
, Yongping You
2
and
Peiyu Pu
1

2
Department of Neurosurgery, Jiangsu Provincial People's Hospital, Nanjing, China ,
3
Shanghai Sensichip Co Ltd,
Shanghai, China,
4
Department of Neurosurgery, Tiantan Hospital, Capital Medical University, Beijing, China


158
Rindflesch et al., 2000; Sekimizu et al., 1998). As no searchable records are available to
efficiently retrieve information relevant to molecular network in glioma, we extracted
glioma-related genes and miRNAs by data mining Pubmed abstracts and established glioma
associated network based on these genes and miRNAs to identify key signalings and
miRNA regulatory module in glioma.
2. Results
2.1 Identification of glioma-related genes and miRNAs
For glioma we queried Pubmed with: glioma[title] AND ("1980/01/01"[PDAT] :
"2010/04/01"[PDAT]). It led to the identification a total of 670 genes and 14 miRNAs that
interacted with glioma. The top 10 glioma-related genes were listed in Table 1. These 14
glioma-related miRNAs were miR-21, miR-34a, miR-221, miR-222, miR-10b, miR-125b, miR-
128, miR-146b, miR-15b, miR-181a, miR-196a, miR-26a, miR-451 and miR-9. Additionally,
we score the journals describing these genes and miRNAs, and the top 10 journals were
listed in Table 2.

Gene PubMed Count
EGFR 130
VEGF 123
GFAP 87
TRAIL 71
CD95 52
JNK 52
ERK 49
IFN 48
PTEN 47
NGF 47
Table 1. The top 10 glioma-related genes.

Journal Count
Cancer Res. 133
J. Neurooncol. 103
Oncogene 53
J. Neurochem. 46
J. Neurosurg. 38
Int. J. Cancer 37
J. Biol. Chem. 36
Biochem. Biophys. Res. Commun. 33
Clin. Cancer Res. 33
Table 2. The top 10 journals describing glioma-related genes and miRNAs.

Data Mining Pubmed Identifies Core Signalings and miRNA Regulatory Module in Glioma

159
2.2 Biological function of glioma-related genes
To better understand the biological role of glioma-related genes, the catalogued genes were
visualized using Gene Ontology (GO) terms and pathway analysis. The Gene Ontology
(GO) provides a structured and controlled ontology for describing gene products in terms of
their associated molecular function, biological process, or cellular component in a species-
independent manner. The molecular function enrichment revealed that 22 GO terms
appeared to be significantly enriched and most glioma-associated genes encode for protein
binding. In the biological process categorythe genes mainly participated in signaling
transduction, response stress, cell differentiation and regulation of cell proliferation. Finally,
the cellular component category found that products of these genes were active mainly in
cytoplasm membrane (Table 3).

Category GO term
molecular
function
protein binding, protein dimerization activity, signal transducer
activity, cytokine activity, enzyme binding, growth factor activity,
growth factor binding, receptor activity, protein kinase activity,
glycosaminoglycan binding, kinase binding, enzyme regulator activity,
G-protein-coupled receptor binding, carbohydrate binding, peptide
receptor activity, collagen binding, polysaccharide binding, kinase
activity, ATP binding, enzyme inhibitor activity, transmembrane
receptor protein tyrosine kinase activity, adenyl nucleotide binding
biological
process
response to stress, regulation of cell proliferation, cell differentiation,
regulation of phosphorylation, regulation of immune system process,
negative regulation of cell proliferation, cell proliferation, anti-
apoptosis, apoptosis, neurogenesis, response to hormone stimulus,
hemopoiesis, regulation of protein kinase activity, immune response,
signal transduction, inflammatory response, cell migration,
angiogenesis, cell communication, phosphorylation, gliogenesis, cell
cycle process
cellular
component
intrinsic to plasma membrane, integral to plasma membrane, plasma
membrane, extracellular region, cell projection, vesicle, nucleoplasm,
cytosol, cell soma, secretory granule, platelet alpha granule,
transcription factor complex, apical plasma membrane
Over-represented GO terms were identified after multiple testing adjustments (P-value<0.05).
Table 3. Set of GO terms with highly enriched genes.
To further explore the pathway involved in these genes, we searched KEGG database for
their pathway information. 16 pathways whose P-value was less than 0.01 were kept (Table
4). The most top enriched pathway is p53 signaling pathway, including 27 genes and Toll-
like receptor signaling pathway including 33 genes.
2.3 Interaction network of glioma-related genes
To uncover the potential interaction networks or synergistic effects of these glioma-related
genes, we employed each gene set as queries and searched for their interaction partners by


160
accessing the database STRING. STRING integrates different public databases containing
information on direct and indirect functional proteinprotein associations by benchmarking
them against the common reference set, KEGG pathway database. 204 genes had
interactions in the database STRING. We next tried to connect these genes into a network to
identify biologically informative linker genes which were statistically enriched for
connections to member of glioma-related gene list. Figure 1A summarized PIK3CA, PIK3CB
and JAK2 three queries served as "hubs" (label with red circle), which has high connection
and was an indicator for essentialness in a network. Surprisingly, further analysis found that
PIK3CA, PIK3CB and JAK2 were associated with signaling transduction, MAPK pathway,
growth factor, cell apoptosis, cell proliferation, cell adhesion and cell migration (Figure 1B).
Given that PIK3CA and PIK3CB encode the protein PI3K subunit p110 and p110,
respectively, these data suggested that PI3K and JAK2 signalings provided excellent
biomarkers for glioma aggressiveness.

Pathway Count Enrichment P-value
p53 signaling pathway 27 0
Toll-like receptor signaling pathway 33 0
Apoptosis 30 9.80E-10
Cytokine-cytokine receptor interaction 65 2.48E-09
Glioma 23 8.80E-08
MAPK signaling pathway 51 1.24E-06
ErbB signaling pathway 24 1.02E-05
Focal adhesion 40 1.12E-05
Cell cycle 28 1.28E-05
T cell receptor signaling pathway 27 1.47E-05
Adipocytokine signaling pathway 20 3.11E-05
Chemokine signaling pathway 38 3.22E-05
Neurotrophin signaling pathway 29 4.10E-05
VEGF signaling pathway 18 0.003731
Adherens junction 18 0.003731
Over-represented KEGG pathways were identified after multiple testing adjustments (P-value<0.05).
Table 4. Set of signaling pathways with highly enriched genes.


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Fig. 1. Visualization of glioma-related gene interaction network.
(A) Connectivity analysis was performed using the Search Tool for the Retrieval of
Interacting Genes/Proteins (STRING) to generate glioma-related gene knowledge-driven
network, as described in Methods. Analysis revealed PI3KCA, PI3KCB and JAK2 are hub
genes with P-value<0.001, which had an influential role in network stability.
(B) PI3K and JAK2 hub signalings located at the key status of glioma-related gene
knowledge-driven network, and exerted a wide effect on kinds of biological functions and
pathways, including signaling transduction, MAPK pathway, growth factor, cell apoptosis,
cell proliferation, cell adhesion and cell migration. Purple lines correspond to activation,
blue lines to inhibition, and yellow lines to association. Red circles (PI3KCA, PI3KCB and
JAK2) are indicated for hub genes.
2.4 Glioma-related miRNA pathway
Because each miRNA target prediction program uses a different computer-aided algorithm
for prediction, encompassing all these methods will probably produce a more reliable model


162
of target prediction. Thus, a union target gene list of 14 glioma-related miRNAs was
generated from 3 target prediction programs (PicTar, TargetScan and miRanda). To further
explore the signaling pathway in these target genes, pathway analysis was performed. Table
5 showed that p53 signaling pathway, Apoptosis, Focal adhesion, MAPK signaling pathway,
Toll-like receptor signaling pathway and Cell cycle pathways were significantly over-
represented. Actually, these 6 pathways were included in the pathways of glioma-related
genes. These findings imply that glioma-related genes and miRNAs prefer a common set of
signaling pathways.

Pathway Count Enrichment P-value
p53 signaling pathway 19 8.67E-09
Apoptosis 21 2.4E-08
Focal adhesion 26 0.000123
MAPK signaling pathway 30 0.000499
Toll-like receptor signaling pathway 15 0.005773
Cell cycle 16 0.006547
Over-represented KEGG pathways were identified after multiple testing adjustments (P-value<0.05).
Table 5. Set of signaling pathways with highly enriched microRNA targets.
In order to construct the network between glioma-related miRNAs and the signaling
pathway, integrated analysis of the targets of glioma-related miRNAs was performed. This
procedure obtained 6 miRNA-pathway networks. For instance, p53 signaling pathway
network contained 12 miRNAs (miR-21, miR-34a, miR-221, miR-222, et al) and 19 genes
(PTEN, CDK6, BBC3, et al) (Fig.2).

Fig. 2. Visualization of miRNA-p53 pathway network in glioma.


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The network was visualized with Medusa software. Blue quadrangles represent glioma-
related miRNAs. Red circles represent miRNA targets that were overlapped by glioma-
related geges.
3. Discussion
The overall utility of our data mining approach, including the strategy for constructing
interaction networks, is to explore biological mechanisms involved in glioma progression. In
this study, we obtained 670 genes and 14 miRNAs that interacted with glioma and
generated interaction networks from abstract-based text mining. Importantly, our analysis
identified PI3K and JAK2 hub signalings and miRNA regulatory module in glioma.
3.1 Core signalings in glioma
By integration of PubMed text mining, homology prediction, gene neighbor, protein-protein
interaction, gene fusion and other data sources, we constructed glioma-related genes
knowledge-driven network. Further analysis revealed that PI3K and JAK2 hub signalings
that had an influential role in network stability, located at the key status of glioma-related
genes knowledge-driven network. These signaling exerted a wide effect on kinds of
biological functions and pathways, including signaling transduction, MAPK pathway,
growth factor, cell apoptosis, cell proliferation, cell adhesion and cell migration. Further,
integrating GO and pathway analysis, data revealed that proliferation without control and
invasive growth were the essential characteristic of glioma.
PI3Ks are heterodimers comprised of a regulatory subunit (p85) and a catalytic subunit
(p110). Activated receptor tyrosine kinases recruit the PI3 kinase complex to the membrane
via the p85 regulatory subunit, thereby activating the catalytic subunit p110, which then
phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-
trisphosphate (PIP3). PIP3 recruits protein AKT to the plasma membrane where AKT is
phosphorylated at Thr308 and Ser473 (Cheng et al., 2009). A high frequency of mutations in
PIK3CA, the gene encoding the p110alpha subunit of PI3K, was found in glioblastoma
(Gallia et al., 2006; Kita et al., 2007). Our recent data showed that PI3K activity were greatly
increased with the ascending of tumor grade and correlated positively with AKT2
expression (Wang et al., 2010). Activation of PI3K/Akt signaling cascade results in cell
survival and proliferation as well as inhibition of cell apoptosis through regulating
downstream targets. AKT contributes to glioma cell migration and invasion by regulating
the formation of cytoskeleton, influencing adhesion and MMP2/9 expression (Pu et al., 2004;
Zhang et al., 2009a; Zhang et al., 2010d). AKT promotes the cell cycle progression by
suppression of cyclin-dependent kinase inhibitors p21 and p27 and increase of Cyclin D1
(Guillard et al., 2009; Koul et al., 2010; Pu et al., 2006). AKT inhibits cell apoptosis by
inactivation of caspase pathway, and activation of BCL2, NFB and mTOR signaling cascade
(Jiang et al., 2009; Ruano et al., 2008; Zhang et al., 2010d). Further, prosurvival signaling by
PI3K contributes to therapeutic resistance in the setting of established antiglioma therapies.
Several studies have shown that PI3K inhibition sensitizes glioma cells to radiation and
chemical therapy (Opel et al., 2008; Prevo et al., 2008). Additionally, our study recently has
showed that co-suppression of PI3K and AKT exerts significant proliferation and invasion
inhibition effects on glioma cells (Fu et al., 2009). In the current study, we found that is PI3K
is a molecular hub in glioma-related genes knowledge-driven network, and associated with
a wide variety of cell biological functions and signaling pathways. Therefore, it is urgent to
develop novel therapies for targeting PI3K/AKT signaling in glioma treatment.


164
In our case, network analysis also identifies a new candidate hub gene JAK2 in
glioblastoma. JAKs, which have four members, JAK1, JAK2, JAK3 and Tyrosine kinase 2
(Tyk2) in mammals, are non-receptor tyrosine kinases involved in upstream intracellular
signaling pathways that become activated after extracellular ligand binding to a variety of
cytokine and growth-factor receptors (Pesu et al., 2008). JAK2 is known to be able to
phosphorylate members of the signal transducers and activators of the transcription (STAT)
protein family, subsequently leading them to translocate to the nucleus and bind to specific
DNA sequences in the promoters of multiple responsive genes (Ghoreschi et al., 2009; Rane
and Reddy, 2000). STAT family has been reported to be involved in the development of
glioma. Of note, STAT3, is aberrantly activated in human glioblastoma tissues, and this
activation is implicated in controlling critical cellular events thought to be involved in
gliomagenesis, such as cell cycle progression, apoptosis and angiogenesis (Brantley and
Benveniste, 2008). Recently, a glioma-specific regulatory network has revealed the
transcriptional module that activates expression of mesenchymal genes in malignant glioma
and STAT3 is one of key transcription factors necessary in human glioma cells for
mesenchymal transformation (Carro et al., 2010). Additionally, nuclear staining of phospho-
STAT5 is overexpressed in glioma tissues, and cytoplasm staining of STAT5b is markedly
increased in glioblastoma multiforme compared with that in normal brain (Kondyli et al.,
2010; Liang et al., 2009). Reduction of STAT5b inhibits glioma cell growth, cell cycle
progression, invasion and migration through regulation of gene expression, such as Bcl-2,
p21, p27 and VEGF (Liang et al., 2009). As another member of STAT family, STAT1 is up-
regulated in the majority of glioblastomas (Haybaeck et al., 2007). Little evidence exists to
show the mechanism of JAK2 (upstream regulator of STAT family) involved in
glomagenesis. However, data mining analysis displays that JAK2 occupies a core regulatory
node of glioma-related genes knowledge-driven network. These data indicate that
modulation of the mechanism responsible for JAK2 in glioma would help us to elucidate the
development of glioma and inhibition of JAK2/STAT signaling could be used as a new
therapeutic strategy to treatment glioma. The JAK/STAT pathway plays a central role in
principal cell fate decisions, regulating the processes of innate immunity, adaptive
immunity, cell proliferation, differentiation, and apoptosis.
In addition, we found the gene CTNNB1 (encoding -catenin) at the lower right corner of
Fig.1 would warrant further investigation. -catenin and Tcf-4 are the core components of
the canonical Wnt/-catenin/Tcf pathway, which is a crucial factor in the development of
many cancers (MacDonald et al., 2009; Ying and Tao, 2009). -catenin accumulates in the
nucleus, where it interacts with coregulators of transcription including Tcf-4 and Lef-1 to
form a -catenin/Tcf/Lef complex. This complex regulates transcription of multiple genes
involved in cellular proliferation, differentiation, survival and apoptosis, including Fra-1, c-
myc and Cyclin D (Wang et al., 2002; Yochum et al., 2008). Recently several reports have
showed that aberrant activation of Wnt/-catenin/Tcf signaling pathway is an important
contributing factor in gliomas (Liu et al., 2010; Pu et al., 2009; Sareddy et al., 2009). -catenin
and Tcf-4 were up-regulated in glioma tissues in comparison to normal brain tissues.
Knockdown of -catenin by siRNA in human glioma cells inhibited cell proliferation and
invasive ability, induced apoptotic cell death and delayed the tumor growth (Pu et al., 2009).
However, up to now, little direct evidence exists to show the mechanism of -catenin and
Tcf-4 involved in gliomagenesis.
Actually, our data doses not well confirm the update results of The Cancer Genome Atlas
Network (TCGA) (Verhaak et al., 2010). TCGA catalogs recurrent genomic abnormalities in


165
GBM, and describes a gene expression-based molecular classification of GBM into
Proneural, Neural, Classical, and Mesenchymal subtypes. Aberrations and gene expression
of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural
subtypes, respectively. Despite of the differences of two studies, our data showed another
approach to explore the mechanism involved in glioma using existing data.
3.2 MiRNA regulatory module in glioma
miRNAs are a new class of small, non-coding RNAs located in noncoding regions or the
introns of the genome, and regulate gene expression by binding to the 3-untranslated
region (3-UTR) of specific mRNAs. Extensive studies have indicated that miRNAs could
function as oncogenic miRNAs or tumor suppressor miRNAs, playing crucial roles in
carcinogenesis. Expression profiling of glioma has unveiled miRNA signatures that not only
distinguish glioma from normal tissues, but can also differentiate histotypes or molecular
subtypes with altered genetic pathways (Ciafre et al., 2005; Lavon et al., 2010). Our data
mining analysis showed that 6 pathways involved in 14 glioma-related miRNAs, in line
with the pathway analysis of glioma-related genes, indicating that glioma-related genes and
miRNAs exert an effect on a common set of signaling pathways. Moreover, we found that
the pathway regulatory control mediated by miRNAs differs from pathway to pathway and
the targets of a specific miRNA are significantly enriched in multiple pathways. In p53
signaling pathway network, 12 miRNAs and 19 genes are involved. Among these miRNA
and target gene relationships, MDM2, CDK6, CDKN2A and CCNE1 are successfully
identified as direct targets of miR-221, miR-34a, miR-125b and miR-15b, respectively (Kim et
al., 2010; Pogue et al., 2010; Sun et al., 2008; Xia et al., 2009). Our data recently showed that
miR-221 and miR-222 directly modulate PTEN expression via targeting PTEN 3-UTR
(Zhang et al., 2010a). In addition, we have also evidenced that BBC3, also named p53 up-
regulated modulator of apoptosis (PUMA), is a new target of miR-221, consistent with
bioinformatics analysis (Zhang et al., 2010b). Further, a recent publication revealed that
miR-21 can impair p53-mediated apoptosis in response to chemotherapeutic (doxorubicin)-
induced DNA damage, therefore contributing to drug resistance in glioblastoma cells
(Papagiannakopoulos et al., 2008). Thus, modulation of these p53-related targets by miR-21
may potentially explain previous observation that p53 signaling pathway were up-regulated
in response to miR-21 knockdown (Frankel et al., 2008). These exciting results prompt us to
further elucidate the intricacy of the interaction between miRNAs and the signaling
pathway.
In conclusion, using data mining analysis, we construct glioma-related genes knowledge-
driven network and show that PI3K and JAK2 hub signalings are key steps leading to
oncogenesis in glioma, and further propose miRNA regulatory module in glioma. These
data demonstrate the power of data mining strategies as tools for biological discovery and
identify core signalings and miRNA regulatory module in glioma, suggesting that the
application of this strategy to consolidate all existing data for other diseases may yield
important discoveries in disease pathogenesis.
4. Experimental procedures
4.1 Natural language processing (NLP) system
Medline/PubMed is used as information source for bioinformatics text mining. Medline
abstracts were retrieved using National Center for Biotechnology Information (NCBI)


166
PubMed portal. We queried Pubmed with: glioma[title] AND ("1980/01/01"[PDAT] :
"2010/04/01"[PDAT]). All abstracts were downloaded as HTML text without images and
then converted into XML documents. Sentence tokenization was performed with Lingpipe
tools. Subsequent analysis is based on the sentence as the basic units. Gene mentions were
tagged using ABNER (Settles, 2005). To solve the matter of plethora of gene aliases, all gene
mentions were normalized to Entrez gene (http://www.ncbi.nlm.nih.gov/Entrez/) official
gene symbols. Only sentences with glioma, the genes were selected.
In order to test the null hypothesis 'the relationship between glioma and the gene is
random', hypergeometric distribution test was employed. Let N be the total number of
PubMed abstracts and m, n be the number mentions in PubMed for glioma and a related
gene, respectively.
1
0
1 ( | , , )
k
i
p p i n m N
=
=

Where
( ) ( ) ( )
!( )! !( )!
( | , , )
! ! ! ! !
n N n m N m
p i n m N
n i i n m N n m i N

=
+

The "glioma-gene" relations with P-value<0.05 were then summarized and subjected to a
relational database for further analysis.
4.2 Gene ontology analysis
Gene ontology analysis was performed by GSEA Base package of BioConductor
(http://www.bioconductor.org/). The glioma-related genes were performed a gene set
enrichment analysis based on the gene ontology (GO) categories.
4.3 Pathway analysis
Expression Analysis Systematic Explorer (EASE) (Hosack et al., 2003) was used to analyze
KEGG pathways. Over representation of genes in a KEGG pathway is present if a larger
fraction of genes within that pathway is differentially expressed compared with all genes in
the genome. The "glioma-gene" relationships retrieved by our NLP system were filtered by
pathway enrichment analysis. The links between glioma and related genes were then
visualized in Cytoscape software (Cline et al., 2007) (http://www.cytoscape.org/). Genes
were grouped according to pathways. Genes that involves in multiple pathways are
assigned to a single pathway with the smallest enrichment P-value.
4.4 Gene network analysis
Integrating PubMed text mining, homology prediction, gene neighbor, protein-protein
interaction, gene fusion and other data sources through the Search Tool for the Retrieval of
Interacting Genes/Proteins (STRING), we created glioma-related genes knowledge-driven
network (von Mering et al., 2005). Linker genes below a P-value threshold of 0.01 were
identified as "hubs". The results from the search are saved in data files describing links
between two genes and then handled in Medusa software.


167
4.5 Target gene prediction and pathway analysis of miRNAs
Three computational tools: TargetScan v5.1 (http://www.targetscan.org/), miRanda v5
(http://microrna.sanger.ac.uk/), PicTar ver. March 26, 2007 (http://pictar.mdc- berlin.de/)
were utilized to identify miRNA targets in 3'-UTR of genes. The union of these results was
listed for further analysis. These targets were used to analyze KEGG pathways.
4.6 MiRNA-target network analysis
The overlap of target genes of glioma-related miRNAs predicted by computational tools and
glioma related genes derived from NLP analysis was calculated. A bipartite network of
microRNAs and corresponding target genes was constructed. The network was displayed in
separated pathways.
5. Acknowledgments
This work was supported by China National Natural Scientific Fund (30971136, 30872657,
81072078), Tianjin Science and Technology Committee (09JCZDJC17600), Program for New
Century Excellent Talents in University (NCET-07-0615), Jiangsu Province's 333 Key
Talent Foundation (0508RS08).
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Part 4
Sequence Analysis and Evolution

0
Signicance Score of Motifs
in Biological Sequences
Grgory Nuel
Institute for Mathematical Sciences (INSMI), CNRS, Paris
Department of Applied Mathematics (MAP5), University of Paris Descartes
France
1. Introduction
In Bioinformatics, it is common to search biological sequences (DNA, RNA, proteins) for
functional motifs such as cross-over hotspot instigators (chi), restriction sites, regulation
motifs, binding sites, active sites in proteins, etc. (Beaudoing et al., 2000; Brazma et al.,
1998; El Karoui et al., 1999; Frith et al., 2002; Hampson et al., 2002; Karlin et al., 1992;
Leonardo Marino-Ramrez & Landsman, 2004; van Helden et al., 1998). Due to evolution
pressure, functional motifs are likely to be more conserved than non-functional motifs. As
a consequence, it is a natural strategy to search biological sequences for motifs which are
statistically exceptional (ex: over- or under-represented).
Given /a motif of interest (from simple strings to complex regular expressions), a recurrent
question is: how surprising is it to observe n occurrences of /in my dataset . In statistical
terms, this is equivalent to compute the p-value of observation n in respect with a relevant
reference model. More precisely, if X
1:
= X
1
. . . X
is a length random sequence generated

by our reference model, and if N denotes the random number of occurrences of /in X
1:
, for
any n 0 our objective is to compute the signicance score of observation n:
S(n) =
_
+log
10
P(N n) if n E[N]
log
10
P(N n) if n > E[N]
(1)
this score representing the p-value in a decimal log-scale, negative (resp. positive) values
being associated to under- (resp. over-) representation events.
In order to compute such a score for a given motif / and a given dataset, one needs two
essential steps:
1) Counting: count the observed number n of occurrences of motif /in the dataset;
2) Signicance: compute the p-value of observation n with respect to a reference model.
In this chapter, we give all the necessary details to perform these two steps using state of the
art approaches including some unpublished results.
2. Counting motifs
2.1 Biological motifs
We can see on Fig. 1 various examples of the kind of biological motifs we usually deal with in
Bioinformatics. In most cases, these motifs are built from a set of active sequences (putative
9
AGCGG GSTGGTGG CCWGG CTGCAG GAATTC CAYNNNNNRTG
CPRRRGRQTYTRFQTLELEKEFHF...........NHYLTRRRRIEIAHAL.........
CPRRRGRQTYTRFQTLELEKEFHF...........NHYLTRRRRIEIAHAL.........
VSVRKKRKPYSKFQTLELEKEFLF...........NAYVSKQKRWELARNL.........
-----------------LTKYFNK...........QPYPTRREIEKLAASL.........
SGKRRRRGNLPKESVQILRDWLYEhr........yNAYPSEQEKVLLSRQT.........
SKKRRHRTTFTSLQLEELEKVFQK...........THYPDVYVREQLALRT.........
SKKRRHRTTFTSLQLEELEKVFQK...........THYPDVYVREQLALRT.........
H-D-[LIVMFY]-x-H-x-[AG]-x(2)-[NQ]-x-[LIVMFY]
A [ 3 21 25 0 0 24 1 0 ]
C [13 1 0 0 5 0 0 0 ]
G [ 4 0 0 0 0 1 0 2 ]
T [ 5 3 0 25 20 0 24 23 ]
Fig. 1. Various kind of biological motifs. From top to bottom: strings in IUPAC
(Cornish-Bowden, 1985) alphabet (DNA), multiple alignment (proteins), sequence logo
(proteins), consensus pattern (proteins), and frequency matrix (DNA). Various sources
including ReBase (Roberts et al., 2010), PROSITE (Sigrist et al., 2010), and JASPAR databases
(Bryne et al., 2008).
or conrmed by experiments) in the form of a multiple alignment or a frequency matrix from
which can be derived a consensus. This consensus could sometimes be a simple string (ex:
AGCGG the chi site of B. subtilis) but in most cases it is a degenerated pattern (ex: CAYNNNNNRTG a
restriction site in the IUPAC alphabet, PROSITE signatures). In all cases however, it is possible
to consider our biological motif /as a (possibly large) set of strings.
Formally, let / be a nite set of strings over a nite alphabet /. Ex: / = A, C, G, T for
DNA sequences; this is the alphabet we are going to use from now on in our examples. Let
X
1:
= X
1
. . . X
be an observed sequence of length over /. Then the number N(/; X

1:
) of
in Biological Sequences 3

(a) NFA for A

(b) NFA for C

(c) NFA for G

(d) NFA for T

0
1
C
2
T
start
(e) NFA for (C[T)
0
1
C
2
T start
C
T
C
T
(f) NFA for (C[T)
0 1
A
start
2
C
3
T
C
T
C
T
(g) NFA for A(C[T)
0
1
A
4
G
start
2
C
3
T
C
T
C
T
(h) NFA for A(C[T)
[G
Fig. 2. Glushkovs construction for A(C[T)
[G. (a), (b), (c), and (d) are singletons; (e) results
from the union of (b) and (d); (f) results from the Kleenes closure of (e); (g) results from the
concatenation of (a) and (f); (h) results from the union of (g) and (c).
matching positions of /in X
1:
, is dened by
N(/; X
1:
) =
i=1
1
X
1:i
/
/
(2)
/
/being the set of all nite sequences over /ending with one element of J (this notation
will be explained in the next section), and where 1
A
is the indicator function of event A.
In the particular case where / contains no strings that are included into each other (which
is a common assumption), the number N of matching position corresponds exactly to the
number of occurrences. However, there is no need to put any restriction on /as long as we
are interested in the number of matching positions like we do.
Fromnowon, if the sequence X
1:
is observed, we denote by the number of matching positions
by n, and if the sequence X
1:
is random, we simply denote by N the random number of
matching positions.
2.2 Regular languages
Let us denote by /
the set of all nite sequences over /. Any subset / /
is then called
a language over /. We denote by T(/
) the set of all possible languages over /. We denote

by /
the empty sequence, and for the sake of simplicity, the singletons of T(/
) will be
simply denoted by their element. Ex: A instead of A, TGC instead of TGC, instead of .
We dene on these languages three regular operations:
Union ([): for all /
1
, /
2
T(/
), /
1
[/
2
= /
1
/
2
. The neutral element of the binary
operator [ is . Ex: AT, GA[T, GA, TT = AT, T, GA, TT.
175 Significance Score of Motifs in Biological Sequences
Require: remove rst all states that are not reachable from or that cannot reach any element
of T
1: J T, Q T and T T, Q T
2: while J is not empty do
3: select and remove 1 fromJ
4: for all a / do
5: o = q Q, (q, a) 1
6: for all ! T such as ! o ,= and ! o do
7: replace !in T by !
1
! o and !
2
! !
1
8: if ! J then
9: replace !in T by !
1
and !
2
10: else
11: if [!
1
[ [!
2
[ then add !
1
to J else add !
2
to J end if
12: end if
13: end for
14: end for
15: end while
Algorithm 1. Performs Hopcrofts reduction on NFA (/, Q, , T, ). J (working set) and T
(partition set) are two sets of set of NFA states. The resulting complexity is O([Q[ log [Q[).
Concatenation (): for all /
1
, /
2
T(/
), /
1
/
2
= xy, x /
1
, y /
2
. The neutral
element of the binary operator is . For all / T(/
), /
0
= (convention), /
1
= /,
/
2
= / / and the notation extends recursively to /
k
for any k 3. Ex: G, GA AT, T =
GAT, GT, GAAT; G, GA
3
= GGG, GGGA, GGAG, GGAGA, GAGG, GAGGA, GAGAG, GAGAGA. For the
sake of simplicity, is implicitly used when the operator is omitted.. Ex: // means / /.
Kleenes closure (
): For all / T(/
), /
=
k0
/
k
. Ex: AT
=
, AT, ATAT, ATATAT, . . ..
The precedence rule of these operations is: [ (lowest precedence), (associative operator),

(highest precedence). Ex: A[C T
= (A[(C(T
)), TT A[C
G = ((TT A)[((C
) G)).
We call regular expression over / any algebric expression over T(/
) dened from singleton

elements and a nite number of regular operations. The resulting language is called a
regular language. Ex: any nite language is a regular language, /
is a regular language,
(A[C[G[T)
GGATG is a regular language, AG, AAGG, AAAGGG, . . . is not a regular language.

2.3 Non-deterministic nite automaton
A Non-deterministic Finite Automaton (NFA) is dened as a 5-tuple (/, Q, , T, ) where: /is a
nite alphabet, Qis a nite state space, Qis the starting state, T Qis the set of nal states,
and : Q/ T(Q) is the transition function. An element X
1:
/
is accepted by this NFA

if and only if it exists a path from the starting state to one of the nal state that sequentially
use the letters X
1:
in the transitions. More formally, it means that it exists a sequence of states
(ie: elements of Q) q
0
= , q
1
, q
2
. . . , q
1
, q
T such as q
i
(q
i1
, X
i
) for all 1 i . The
language of a NFA is the set of all elements of /
it accepts.
Theorem 1. For any language / T(/
): / regular it exists a NFA whose language is

/.
We admit that the language of a NFA is always regular (see Hopcroft et al., 2001, for the
formal proof) but we will prove the reciprocal with the Glushkovs construction (Allauzen &
Mohri, 2006). This construction provides a simple way to build the NFA directly from the
regular expression of the language. The idea is to treat the regular expression as any algebraic
expression with a stack of operands (NFAs) and a stack of operators (regular operations).
Since a regular expression is by denition built from singleton elements of /
and the three

regular operations, we only need to give the construction of a NFA corresponding to singleton
elements, and the constructions corresponding to the regular operations.
Singleton: for any X
1:
/
we build the NFA (/, Q, , T, ) with Q = 0, 1, . . . , , = 0,

T = , and (i 1, X
i
) = i for all 1 i .
Union: the union (/, Q, , T, ) of two NFAs (/, Q
1
,
1
, T
1
,
1
) and (/, Q,
2
, T
2
,
2
) is given
by: Q = Q
1
Q
2

2
, =
1
, T = T
1
T
2
and
(q, a) =
_
1
(
1
, a)
1
(
2
, a) if q =
1
1
(q, a) if q Q
1

1
2
(q, a) if q Q
2

2
. (3)
Concatenation: the concatenation (/, Q, , T, ) of two NFAs (/, Q
1
,
1
, T
1
,
1
) and
(/, Q,
2
, T
2
,
2
) is given by: Q = Q
1
Q
2

2
, =
1
, T = T
2
and
(q, a) =
_
1
(q, a) if q Q
1
T
1
2
(
2
, a) if q T
1
2
(q, a) if q Q
2

2
. (4)
Kleenes closure: the Kleenes closure (/, Q, , T, ) of NFA (/, Q
1
,
1
, T
1
,
1
) is given by:
Q = Q
1
, =
1
, T = T
1

1
and
(q, a) =
_
1
(q, a) if q Q
1
T
1
1
(
1
, a) if q T
1
. (5)
Using Glushkovs construction, it is then possible to build a NFA whose language correspond
to the regular expression of our choice. However in general, this construction is not optimal
in terms of number of states. Fortunately, the reduction algorithm (Algorithm 1) due to
Hopcroft provides a (partial) solution to this problem. Note that nding a minimal NFA
for a given regular expression is a difcult task in general, but that Hopcrofts reduction is
a good heuristic (we will see later that in the case of DFA, Hopcrofts reduction is indeed a
minimization).
2.4 Counting with NFA
NFAs provide with Algorithm 2 an extremely efcient way to look for matching positions
of any motif / (in fact, any regular expression) in a sequence X
1:
. The algorithm directly
results from the denition of the language of a NFA.
Let us illustrate this algorithm with a toy example: how to nd all matching positions of / =
G(G[C)G in X
1:12
= AGCGGTGGGCGA ? We rst use Glushkovs construction and Algorithm 1 to
obtain on Fig. 3 a minimal NFA whose language is (A[C[G[T)
G(G[C)G. Then we directly apply

Algorithm 2 starting with o = 0:
i = 1, X
1
= A, o (0, A) = 0;
i = 2, X
2
= G, o (0, G) = 0, 1;
Require: (/, Q, , T, ) be a (minimal) NFA whose language is /
/
1: o
2: for i = 1 . . . do
3: o
qo
(q, X
i
)
4: if o T ,= then
5: report i as a matching position
6: end if
7: end for
Algorithm 2. NFA pattern matching. Returns all matching positions of motif /in X
1:
.
Complexity is O([Q[ ).
0
A,C,G,T
1
G
start 2
G,C
3
G
Fig. 3. Minimal NFA whose language is (A[C[G[T)
G(G[C)G.
i = 3, X
3
= C, o (0, 1, C) = 0, 2;
i = 4, X
4
= G, o (0, 2, G) = 0, 1, 3, matching position;
i = 5, X
5
= G, o (0, 1, 3, G) = 0, 1, 2;
i = 6, X
6
= T, o (0, 1, 2, T) = 0;
i = 7, X
7
= G, o (0, G) = 0, 1;
i = 8, X
8
= G, o (0, 1, G) = 0, 1, 2;
i = 9, X
9
= G, o (0, 1, 2, G) = 0, 1, 2, 3, matching position;
i = 10, X
10
= C, o (0, 1, 2, 3, C) = 0, 2.
i = 11, X
11
= G, o (0, 2, G) = 0, 1, 3, matching position;
i = 12, X
12
= A, o (0, 1, 3, A) = 0.
We hence return three matching positions: 4, 9 and 11.
One should note in this example that in twice occasions, we need to recompute a previously
computed transition (i = 7 and i = 11). Obviously, this kind of event is likely to appear
very often when working with longer sequences. It is hence a natural idea to store in memory
previously computed transitions. This approach, known as lazy determinization (Green et al.,
2004), speeds up considerably pattern matching (reducing the complexity from O([Q[ ) to
O()) at the expense of a higher memory usage. We will see later that the amount of memory
needed can increase exponentially with the NFA size [Q[; this problem is usually addressed
by allocating a xed amount of memory to a buffer of computed transitions which is ushed
when full.
3. Signicance
Since we now have efcient algorithms to count the number of occurrence of a motif / in a
sequence X
1:
, let us deal with the signicance of an observation n.
3.1 Reference model
The choice of a reference model is obviously a key point. Since biological sequences like DNA
or proteins are known to have unbalanced letter compositions, it is hence clear that our model
should at least take into account this source of bias. A natural parametric approach
1
is hence
to model X
1:
as a i.i.d. sequence with P(X
i
= a) = (a) a / with all (a) [0, 1] and
a/
(a) = 1. This model is called model M0 with parameter .
For example, in the complete genome of HIV1 (Genbank AF033819) we observe the following
counts: 3272 A, 1642 C, 2225 G, and 2042 T. The maximum likelihood estimates of a M0 model
based on this observation is then: (A) = 3272/9181 35.64%, (C) = 1642/9181 17.88%,
(G) = 2225/9181 24.23%, and (T) = 2042/9181 22.24%.
But if we look now to the frequencies of di-nucleotides on the same HIV1 genome, we observe
considerable bias as well:
AA 1087 AC 524 AG 971 AT 690
CA 754 CC 378 CG 82 CT 427
GA 769 GC 425 GG 625 GT 406
TA 662 TC 315 TG 546 TT 519
For example, we observe 971/3272 = 29.68% of G after a A, but a G occurs after a C only
82/1641 = 16.41% of the time. This phenomenon is directly explained by the fact that the
di-nucleotide CG tend to be easily methylated (see CpG island, Fatemi et al., 2005). Is hence
tempting to take into account the frequencies of di-nucleotides in our reference model, or
tri-nucleotides, or more, which naturally leads to Markov models.
For any d 0, we denote by Md the (homogeneous) Markov model of order d dened for any
i d +1, a /
d
, and b / by:
P(X
i
= b[X
id:i1
= a) = (a, b) (6)
where denotes the transition matrix of Md. This model is clearly dened conditionally to
X
1:d
.
The maximum likelihood estimator is then given for all a /
d
, and b / by:
(a, b) =
n
ab
b
/
/
n
ab
/
(7)
where n
ab
are the observed counts of word ab in the training dataset.
When working with Markov model and biological sequences, a recurrent question is: what
order d should I choose for my reference model ? This is a classical model selection problem
which can easily be solved using penalized likelihood criteria like BIC or AIC (Liddle, 2007).
For example, using the BIC criterion, one would select d = 1 for the complete genome of
HIV1 ( 10kb), and d = 5 for the complete genome of E. coli ( 4.6Mb). However, since
our objective is the signicance of motifs counts rather than the modelization of biological
sequence in itself, we suggest a different approach.
First, it is critical to realize than working with a model Md as reference model allows to take
into account the sequence composition bias in (d +1)-mers. Hence, with d = 1 one takes into
account the composition bias in di-nucleotides, and with d = 5, one takes into account the
composition bias in hexa-nucleotides. The decision could then be based on the information
one wishes to include in the reference model; working on coding sequences, one might wish
to take into account at least the codon bias hence resulting in the choice of d 2. On the other
1
An alternative non-parametric approach, the shufing, consists in performing uniformly a random
permutation of the original sequence; this approach is not treated here.
Require: (/, Q
1
, , T
1
,
1
) a NFA
1: q
0
, L 1, Q
2
q
0
, T
2

2: for i = 0 . . . L 1 do
3: for all a / do
4: o
1
(q
i
, a)
5: if j, q
j
= o then
6:
2
(q
i
, a) = q
j
7: else
8: q
L
o, L L +1, Q
2
Q
2
q
L
9: if o T
then T
2
T
2
q
L
end if
10: end if
11: end for
12: end for
Output: return (/, Q
2
, q
0
, T
2
,
2
)
Algorithm 3. Determinization. Build a DFA which recognizes the same language than the
original NFA.
hand, it would obviously be pointless to use a reference model of order d = 7 to study a motif
of length 8 or less.
Another critical point to keep in mind is that motif signicance is by nature very sensitive to
the parameters of the reference model. In order to convince us, let us a consider the following
simple example with / = GGATG, a reference model M0 of parameter , and = 1, 000, 000. If
(A) = (T) = 0.10 and (C) = (G) = 0.40 we get E[N
] = 0.40
3
0.10
2
640.0. Now
if (A) = (T) = 0.08 and (C) = (G) = 0.42 then E[N
] = 0.42
3
0.08
2
474.2. If we
admit that the standard deviation of N
is roughly equal to = 25 (we will see later on how

to perform such computation), an observation of n = 550 could be interpreted as a signicant
over-representation with the rst parameters, and a signicant under-representation with the
second parameters (observation n deviates from the expectation by more than three standard
deviations in both cases). The reason behind this is that parameter values are typically
involved in complex products when evaluating the signicance of an observation, and that
such operations usually increase small variations rather than averaging them(like with sums).
This problem have been investigated in Nuel (2006c) where it is shown that unwise choices of
d might lead to many false positive results.
3.2 Monte-Carlo simulations
Since the theoretical distribution of N not easy to obtain, it is tempting to study it from
the empirical point of view by performing simple simulations. The approach is quite
straightforward:
1) generate a random dataset i according to the reference model;
2) count the number of occurrence n
i
of /in the dataset;
3) repeat 1) and 2) until we have a sample n
1
, n
2
, . . . , n
r
.
Once a reference sample have been obtained, we can derive the empirical p-value of the
observation n using:
P(N n) =

r
i=1
1
n
i
n
r
or

P(N n) =

r
i=1
1
n
i
n
r
(8)
start 0
A,C,T
1
G
A,T
2
C
3
G
A,C,T
4
G
A,T
C
5
G
A,T
C
G
A,T
C
G
Fig. 4. Minimal DFA whose language is (A[C[G[T)
G(G[C)G.
or, alternatively, one might use this sample to derive empirical expectation, variance, and
z-score:
Z(n) =
n
with =
1
r
r
i=1
n
i
and
2
=
1
r
r
i=1
(n
i
)
2
. (9)
If this approach is quite simple, it suffers several drawbacks: 1) it is slow; 2) sample size must
be large to obtain accurate results. Indeed, if the true p-value is p, then p B(r, p) where r
is the sample size. The following table gives a 90% upper bound condence for p for several
value of r in the case where p = 10
5
:
r 10
3
10
4
10
5
10
6
10
7
10
8
bound 1.00 10
3
1.00 10
4
3.00 10
5
1.50 10
5
1.14 10
5
1.04 10
5
we clearly see that it requires at least r = 10
6
samples to obtain the rst accurate digit in p,
and a prohibitive r = 10
8
samples for the second digit. Considering that very small p-value
are easily encountered in motif signicance (ex: 10
20
, 10
50
, 10
100
), it is clear that empirical
p-value have a limited interest in this context.
Empirical z-score does not suffer the same drawback but makes the implicit assumption that
N has a Gaussian distribution which is highly questionable as we will see later on.
For completeness, let us point out that importance sampling techniques might solve the
estimation problem by sampling N using a tailored dataset distribution (Chan et al., 2010).
However, these sophisticated numerical techniques are slow and requires a good skills to be
implemented.
3.3 Markov chain embedding
The key to perform any motif signicance computation if rst to embed the original problem
into an order 1 Markov chain taking into account all the combinatoric complexity. This
technique, called Markov chain embedding have been used by many authors in the context
of motif signicance Antzoulakos (2001); Boeva et al. (2005); Chang (2005); Fu (1996); Nuel
(2006a), but it is only recently that its connexion to NFA and Deterministic Finite Automata
(DFA) have been pointed out (Crochemore & Stefanov, 2003; Lladser, 2007; Nicodme et al.,
2002; Nuel, 2008a; Nuel & Prum, 2007; Ribeca & Raineri, 2008).
We start with a NFA whose language is /
/ from which we build a DFA (/, Q, q

0
, T, )
using the determinization algorithm (Algorithm 3). A DFA differs from an NFA only by the
denition of its transition function: : Q / T(Q) for a NFA, and : Q / Q
for a DFA. For example, we can see on Figure 4, a (minimal) DFA whose language is
(A[C[G[T)
G(G[C)G. This DFA has more states (6) than the corresponding NFA (4). In fact,
since the state space Q
2
of a DFA corresponds to a subset of the parts of the original NFA state
space Q
1
, we have [Q
2
[ 2
[Q
1
[
. Fortunately, this upper bound is seldom reached in practice.
Theorem 2 (Markov chain embedding for Model M0). Let (/, Q, , T, ) be a (minimal)
DFA whose language is /
/. Let X
1:
be a random sequence generated by the M0 model
of parameter . We consider the sequence Z
0:
recursively dened by Z
0
= , and Z
i
=
(Z
i1
, X
i
) for all 1 i . Then Z
0:
is an order 1 Markov chain whose transition matrix T
is dened for all p, q Q by:
T(p, q) =

a/,(p,a)=q
(a) (10)
and having the following property for all 1 i : X
1:i
/
/ Z
i
T.
For example, if we consider the DNA motif G(G[C)G and the corresponding DFA of Figure 4,
we get the following transition matrix:
T =
_
_
_
_
_
_
_
(A) +(C) +(T) (G) 0 0 0 0
(A) +(T) 0 (C) (G) 0 0
(A) +(C) +(T) 0 0 0 (G) 0
(A) +(T) 0 (C) 0 0 (G)
(A) +(T) 0 (C) (G) 0 0
(A) +(T) 0 (C) 0 0 (G)
_
_
_
_
_
_
_
.
In order to extend Theorem 2 to order Md with d > 0 it is necessary to build DFA
(/, Q, , T, ) be a (minimal) DFA whose language is /
/ and with the property that for

all q Q, past(q) = a /
d
, p Q, (p, a) = q is either empty or a singleton. A DFA
having this property is called a order d DFA by Lladser (2007), and is called non d-ambiguous
by Nuel (2008a). The construction of such a (minimal) DFA is not very complicated but is a
bit technical. A possible approach suggested by Nuel (2008a) consists in starting from a DFA
without this property and duplicating any "ambiguous" state. Another more straightforward
approach consists in adding the elements of /
/
d
to the original language with a specic
label for the nal states corresponding to each elements of /
d
, and to keep these labels during
minimization and determinization algorithms.
Theorem3 (Markov chain embedding for Model Md). Let (/, Q, , T, ) be a (minimal) order
d DFA whose language is /
/. Let X
1:
be a random sequence generated by the Md model
of parameter . We consider the sequence Z
d:
recursively dened by Z
d
= (, X
1:d
), and
Z
i
= (Z
i1
, X
i
) for all 1 i . Then Z
d:
is an order 1 Markov chain whose transition
matrix T is dened for all p, q Q by:
T(p, q) =

a/,(p,a)=q
(past(p), a) (11)
and having the following property for all 1 i : X
1:i
/
/ Z
i
T.
One should note that Z
d:
is dened on (, /
d
/
) which could be slightly smaller than Q.

This subset corresponds to the states of Q having a order d past. If we consider the DFA
start 0
1:A
A
2:C
C
3:G
G
4:T
T
A
C
G
T
A
C
G
T
A
T
5:C
C
6:G
G
A C
G
T
A
C
T
7:G
G
A
T
C
8:G
G
A
T
C
G
A
T C
G
Fig. 5. Minimal order 1 DFA whose language is (A[C[G[T)
G(G[C)G. The order 1 past of each

state is indicated in the state itself. Diamond-shaped states correspond to the elements of
(0, /
1
).
of Figure 5, d = 1, and with X
1
= A, we see that the Markov chain Z
d:
is dened on
1, 2, 3, 4, 5, 6, 7, 8 by Z
1
= 1 and the following transition matrix:
T =
_
_
_
_
_
_
_
_
_
_
_
(A, A) (A, C) (A, G) (A, T) 0 0 0 0
(C, A) (C, C) (C, G) (C, T) 0 0 0 0
(G, A) 0 0 (G, T) (G, C) (G, G) 0 0
(T, A) (T, C) (T, G) (T, T) 0 0 0 0
(C, A) (C, C) 0 (C, T) 0 0 (C, G) 0
(G, A) 0 0 (G, T) (G, C) 0 0 (G, G)
(G, A) 0 0 (G, T) (G, C) (G, G) 0 0
(G, A) 0 0 (G, T) (G, C) 0 0 (G, G)
_
_
_
_
_
_
_
_
_
_
_
.
From now on, we assume that our motif problem with Md reference model is embedded into
the Markov chain Z
d:
whose transition matrix is decomposed into T = P +Q where matrices
P and Q are dened for all p, q by: P(p, q) = T(p, q)1
q/ T
, and Q(p, q) = T(p, q)1
qT
.
3.4 Main results
We present here the main results that are then used to derive exact computations and various
approximations of S(n). In all this section, we assume that N is the random number of
occurrences of /in X
1:
, a sequence generated by a Md model (X
1:d
being xed) with d 0.
we denote by T = P + Q be the transition (L L) matrix of the Markov chain embedding of
the corresponding problem. We also introduce two vectors: u a 1 L vector lled with 0 and
having a 1 in the position corresponding to X
1:d
, and v a L 1 vector of 1.
Proposition 4 (probability generating function). If we denote by G(y) = E[y
N
] the probability
generating function (pgf) of N, then we have:
G(y) =

n0
P(N = n)y
n
= u(P + yQ)
d
v. (12)
Proof. The rst equality derives directly from the denition of G(y). For the second equality
now, it is clear that u(P + Q)
d
gives the marginal distribution of Z
. We then connect this

distribution to N by counting the number of times we use the transitions of Qwith the dummy
variable y so that u(P + yQ)
d
gives the joint distribution of (Z
, N). Finally, we sum up the

contributions of all states using the product with v.
For example, let us consider / = G(G[C)G and X
1:12
generated by a M0 model with
parameters (A) = (T) = 0.10 and (C) = (G) = 0.40. Proposition 4 hence gives:
G(y) =
_
1 0 0 0 0 0
_
_
_
_
_
_
_
_
0.6 0.4 0 0 0 0
0.2 0 0.4 0.4 0 0
0.6 0 0 0 0.4y 0
0.2 0 0.4 0 0 0.4y
0.2 0 0.4 0.4 0 0
0.2 0 0.4 0 0 0.4y
_
_
_
_
_
_
_
12
_
_
_
_
_
_
_
1
1
1
1
1
1
_
_
_
_
_
_
_
(13)
= 0.33369 +0.31148y +0.19357y
2
+0.09681y
3
+0.04140y
4
+0.01569y
5
+0.00528y
6
+0.00157y
7
+0.00042y
8
+0.00008y
9
+0.00002y
10
. (14)
From this result, we have the whole distribution of N: support is 0, 1, . . . , 10, P(N = 0) =
0.33369, P(N = 1) = 0.31148, . . . , P(N = 10) = 0.00002. We can also easily derive moments
of N from this distribution: E[N] = 1.28, [N] = 1.29.
Lemma 5 (derivatives of the pgf). For any k 0, the order k derivative of the pgf G is given
by:
G
(k)
(y) = k![z
k
]u(P + yQ+ zQ)
d
v (15)
where the [z
k
] operator denotes the extraction of the coefcient of z
k
in the expression.
Proof. The formal proof can be found in Nuel (2010) in a slightly less general case. Here we
prove it only for the rst two derivatives in the particular case where d = 3. Starting from
G(y) = u(P + yQ)
3
v we get:
G
/
(y) = u
_
Q(P + yQ)
2
+ (P + yQ)Q(P + yQ) + (P + yQ)
2
Q
_
v (16)
and
G
//
(y) = 2u
_
Q
2
(P + yQ) +Q(P + yQ)Q+ (P + yQ)Q
2
_
v (17)
which are easily connected to the terms coefcients of z
1
and z
2
in u(P + yQ+ zQ)
d
v.
If we denote for all k 0 the k-th factorial moment of N by F
k
= E[N!/(N k)!], then, by the
denition of the pgf, it is clear that F
k
= G
(k)
(0), and thanks to Lemma 5 we get:
F
k
= k![z
k
]u(T + zQ)
d
v. (18)
And if we now denote the moment generating function (mgf) of N by M(t) = E[e
tN
] = G(e
t
),
and the cumulant generating function (cgf) of N by (t) = logE[e
tN
] = log M(t) = log G(e
t
),
we get directly the k-th moment of N: E[N
k
] = M
(k)
(0); and the k-th cumulant of N:
k
=
(k)
(0).
Corollary 6 (characteristics moments). If we denote by =
1
the expectation of N, by =

2
the standard deviation of N, by
1
=
3
/
3
the skewness of N, and by
1
=
4
/
4
the excess
kurtosis of N, then we get: = F
1
,
2
= F
2
+ F
1
F
2
1
,
1
=
3F
2
3F
2
1
+ F
3
3F
1
F
2
+2F
3
1
+ F
1
3
, (19)
and
2
=
7F
2
7F
2
1
+6F
3
18F
1
F
2
+12F
3
1
+ F
4
4F
1
F
3
3F
2
2
+12F
2
1
F
2
6F
4
1
+ F
1
4
. (20)
Proof. On just need to compute the derivatives
(1)
(0),
(2)
(0),
(3)
(0), and
(4)
(0).
If we consider again / = G(G[C)G and X
1:12
generated by a M0 model with parameters
(A) = (T) = 0.10 and (C) = (G) = 0.40. Eq. (18) hence gives:
k0
F
k
k!
=
_
1 0 0 0 0 0
_
_
_
_
_
_
_
_
0.6 0.4 0 0 0 0
0.2 0 0.4 0.4 0 0
0.6 0 0 0 0.4 +0.4y 0
0.2 0 0.4 0 0 0.4 +0.4y
0.2 0 0.4 0.4 0 0
0.2 0 0.4 0 0 0.4 +0.4y
_
_
_
_
_
_
_
12
_
_
_
_
_
_
_
1
1
1
1
1
1
_
_
_
_
_
_
_
(21)
= 1 +1.28y +1.01683y
2
+0.61211y
3
+0.29709y
4
+0.11835y
5
+0.03845y
6
+0.00992y
7
+0.00193y
8
+0.00025y
9
+0.00002y
10
. (22)
From this result, we can get all factorial moments of N: E(1) = F
0
= 1, E(N) = F
1
= 1.28,
E(N(N 1)) = F
2
= 2.033664, E(N(N 1)(N 2)) = F
3
= 3.6726374, E(N(N 1)(N
2)(N 3)) = F
4
= 7.1302266, . . . , E(N!/(N 10)!) = F
10
= 60.881161. Thanks to Corollary 6
we get the following characteristic moments: = 1.28, = 1.294320,
1
= 1.163783,
2
=
1.492661.
3.5 Exact computations
As we have seen above, Proposition 4 provides a way to obtain the whole distribution of N
by computing G(y) = u(P + yQ)
d
v from which we can easily derive S(n) for any n 0:
S(n) =
_
_
_
+log
10
_
n
k=0
[y
k
]G(y)
_
if n E[N]
log
10
_
+
k=n
[y
k
]G(y)
_
if n > E[N]
.
From the algorithmic point of view, there are basically two approaches to compute S(n) using
Expression (12). The rst one, called power, consists in computing (P + yQ)
d
using the
power method and a binary decomposition of d. Ex: if d = 1097 then d =
2
10
+ 2
6
+ 2
3
+ 2
0
. We then just have to recursively compute D
k
(y) = (P + yQ)
2
k
using the
relation D
k+1
(y) = D
k
(y) D
k
(y) for all k 0. Since in the computation of S(n) we are only
interested in terms of degree n or less (or n or more), we can easily truncate
2
all polynomials at
degree n thus dramatically reducing the computational costs of polynomial products. We end
2
In the case of over-representation, all contributions of degree n or more are summed into the term of
degree n.
up with a O(log
2
n
2
L
3
) complexity in time where L is the order of the transition matrix
T = P+Q. The corresponding memory complexity is O(log
2
n L
2
). Since the length of
the dataset appears in a logarithmic scale in these complexity, the power approach is obviously
suitable for large datasets (ex: = 10
6
or = 10
9
). Unfortunately, the cubic complexity
with L (quadratic in memory) prevents the approach to deal with complex motifs with high
L. One should also note that the quadratic complexity in n could really be a problem when
dealing with frequent motifs and/or large datasets. In order to overcome this problem, Ribeca
& Raineri (2008) suggested to use fast Fourier transforms (FFT) to perform all polynomial
product hence replacing n
2
by n log
2
n in the time complexity. However appealing at rst
glance, this approach is not recommended in practice since the FFT products in oating-point
arithmetics induce numerical instabilities that make totally unreliable the smallest coefcients
of the polynomials. And unfortunately, these coefcients are precisely the one needed to study
the tail distribution of N.
Another interesting approach called full recursion, consists in computing v
i
= (P + yQ)
i
v for
all 0 i d recursively using the relation v
i+1
= (P+yQ)v
i
. There are two main interests
for this approach: 1) we have only products between polynomials of degree 1 and polynomials
of degree n (by dropping terms of degree greater than n like in the power approach); 2) we can
take full advantage of the sparse structure (only L [/[ non-zero terms in the worst case) of
the transition matrix T = P + Q. The resulting complexity is O( L [/[ n) in time, and
O(L n) in memory. Since these complexities are linear with L, this approach is able to handle
very complex motifs. The drawback is that the approach can be very slow when dealing with
large and n. It exists a sophisticated version of this recursion called partial recursion (see Nuel
& Dumas, 2010) which allows to replace n by log n
2
in the time complexity. However,
the quadratic complexity in n and numerical instabilities in oating-point arithmetic restrains
its use to small n (ex: n 10).
For completeness, let us point out another approach to the problem. The idea is that we can
derive from Expression (12) the following expression:
G(y, z) =

n0
d
P(N
= n)y
n
z
= uz
d
(I Pz + yzQ)
1
v (23)
where I is the identity matrix and N
the number of matching position in X

1:
. It is then
possible to obtain P(N
= n) for any and n using (fast) Taylor expansions of G(y, z). For the
mathematician, this approach is so natural that it is often referred as the golden approach
to the problem of motif signicance (Nicodme et al., 2002). However, this approach suffers
several severe drawbacks that dramatically limits its practical interest: 1) the approach needs
sophisticated computer algebra systems to be implemented (rather than simple oating point
arithmetic for the previous approaches); 2) the explicit computation of (I Pz +yzQ)
1
could
be very time (and memory) consuming; 3) even if the explicit computation of the inverse
matrix is avoided (which is highly advisable), the coefcient extraction using state of the
art techniques (like high-order lifting for example) is often slower than the much simpler
alternative developed above (see Nuel & Dumas, 2010, for details).
Considering either the power or the recursion approaches we obtain easy to implement
algorithms allowing to compute the exact value of S(n) in all cases except when dealing with
high complexity motifs (large L) and/or frequent motifs (large n). But even if we stick to
more tractable cases, exact computations could be slow. The question hence is: is it possible
to compute fast and reliable approximations of S(n) ?
expectation std. dev. skewness e. kurtosis time (s)
12 1.280000 1.294320 1.163783 1.492661 0.01
120 15.104000 4.585724 0.361328 0.149974 0.02
1200 153.344000 14.648033 0.113920 0.014936 0.03
12000 1535.744000 46.367282 0.036014 0.001492 0.04
120000 15359.744000 146.640798 0.011394 0.000410 0.05
Table 1. Characteristic moments the number N of occurrences of motif / = G(G[C)G in a
sequence X
1:
generated by a M0 model with parameters (A) = (T) = 0.10 and
(C) = (G) = 0.40. Computation performed using the power approach.
3.6 Near-Gaussian approximations
Since the random count N is basically dened by Eq. (2) as large sum of Bernouilli variables,
the idea of approximating the distribution of N using Gaussian approximation sounds
appealing. Indeed, Gaussian approximations are historically the rst ones to have been
suggested for this problem (Cowan, 1991; Kleffe & Borodovski, 1997; Pevzner et al., 1989;
Prum et al., 1995). From the theoretical point of view, Central Limit Theorems (CLT) for
weakly dependent variables ensure that N is asymptotically normal distributed. On Table 1,
we can see the characteristic moments of N for motif / = G(G[C)G and various value of the
sequence lengths . According to theory, we observe that the skewness and excess kurtosis
both decease toward 0 when grows (a normal distribution has null skewness and excess
kurtosis). But it is also clear that N is not normally distributed for small values of . As a
consequence, the quality of a Gaussian approximation for S(n) is expected to be questionable
at nite distance.
In order to overcome this issue, Nuel (2010) suggested to consider near Gaussian
approximations instead of simple Gaussian approximations for this problem. The idea is
simply to perform a higher order asymptotic development that exploits more than the two
rst moments of N. This technique is known as the Edgeworths expansion. Blinnikov &
Moessner (1998) gives a general (and rather complicated) formula for this expansion. For
practical purpose, we present the result only up to order 3 expansions.
Proposition 7 (Edgeworths expansion). If we denote by (z) = exp(z
2
/2)/
2 the
probability distribution function (pdf) of a standard Gaussian, then for all n 0 we have
the following approximation:
P(N = n)
(z)
_
C
0
(z) +C
1
(z) +
2
C
2
(z) +
3
C
3
(z)
_
(24)
with
C
0
(z) = 1 C
1
(z) =
S
3
6
H
3
(z) C
2
(z) =
S
4
24
H
4
(z) +
S
2
3
72
H
6
(z) (25)
C
3
(z) =
S
5
120
H
5
(z) +
S
3
S
4
144
H
7
(z) +
S
3
3
1296
H
9
(z) (26)
where = E[N], =
_
V[N], z = (n )/, S
k
=
k
/
2k2
for all k 1, and where H
k
(z)
are the Hermite polynomials dened recursively by H
0
(z) = 1 and H
k
(z) = zH
k1
(z)
H
/
k1
(z) for all k 1.
50 100 150 200 250 300
1
0
1
2
3
4
5
n
e
r
r
o
r
order 0
order 1
order 2
order 3
(a) Error
50 100 150 200 250 300
0
2
4
6
8
n
l
o
g
1
0
(
r
e
l
a
t
i
v
e

e
r
r
o
r
)
order 0
order 1
order 2
order 3
(b) Relative error
Fig. 6. Reliability of NG approximations for / = G(G[C)G on a random sequence X
1:
generated by a M0 model with parameters (A) = (T) = 0.10 and (C) = (G) = 0.40, and
with = 1200. The error NG
h
(n) S(n) is given on Figure (a); and the relative error
(log-scale) log
10
[NG
h
(n) S(n)[/[S(n)[ on Figure (b). The horizontal rule indicates the
null error on Figure (a), and the threshold corresponding to two correct digits on Figure (b).
For h 0, 1, 2, 3 we dene the Near Gaussian (NG) approximation of order h of S(n) by:
NG
h
(n) =
_
_
+log
10
_
_
n
k=0
1
_
k
_
h
j=0
j
C
j
_
k
_
_
_
if n E[N]
log
10
_
_
+
k=n
1
_
k
_
h
j=0
j
C
j
_
k
_
_
_
if n > E[N]
. (27)
We can see on Figure 6 the reliability of NG approximations. In solid black, the order 0
approximation corresponds to the classical Gaussian approximation. Unsurprisingly, this
central limit approximation is accurate for the center of the distribution (n close to the
expectation = 153.3), the reliability quickly deceases when [n [ increases.
Central limit theorems (CLT) for N have established long ago that N should be asymptotically
Gaussian distributed. The problem however with CLT theorems is that the quality of
the resulting approximation dramatically decreases at nite distance when considering tail
distribution events. Here we try to overcome the issue by considering Near-Gaussian
approximations that exploits higher moments of N to improve the quality of the
approximations. In order to do this, a critical problem is rst to obtain the rst k-th moments
of N. Of course we can access these moments by computing the full distribution of N, but
if it is possible to do so, why bothering with approximations. We hence need an method to
compute the moments of N whose complexity should be somehow signicantly smaller than
the complete exact computations. With higher order approximation, we can see a dramatic
improvement of reliability of the results, with a noticeable increase of the region where at
least two digits are correct (up to n [80; 240] for NG
3
).
0 100 200 300 400
1
.
5
1
.
0
0
.
5
0
.
0
0
.
5
1
.
0
1
.
5
n
e
r
r
o
r
Cramer
BahadurRao
(a) Error
0 100 200 300 400
1
0
1
2
3
4
5
n
l
o
g
1
0
(
r
e
l
a
t
i
v
e

e
r
r
o
r
)
Cramer
BahadurRao
(b) Relative error
Fig. 7. Reliability of CB and BR approximations for / = G(G[C)G on a random sequence X
1:
generated by a M0 model with parameters (A) = (T) = 0.10 and (C) = (G) = 0.40, and
with = 1200. The error CB(n) S(n) or BR(n) S(n) is given on Figure (a); and the
relative error (log-scale) log
10
[CB(n) S(n)[/[S(n)[ or log
10
[BR(n) S(n)[/[S(n)[ on
Figure (b). The horizontal rule indicates the null error on Figure (a), and the threshold
corresponding to two correct digits on Figure (b).
From the computational point of view, the order h approximation requires the cumulants of
N up to order h + 2. Using the power approach, the resulting complexity is hence O(log
2

h
2
L
3
) in time and O(log
2
(h +2) L
2
) in memory. Using the recursion, the complexity
resulting complexity is O( L [/[ h) in time, and O(L h) in memory. In both cases,
the computational time drops signicantly from the exact computations.
Thanks to NG approximations, we hence have a fast and reliable way to compute an
approximation of S(n) when n falls in the center of the distribution (ex: [S(n)[ 3.0), but
NG approximations unfortunately remain totally unreliable for tail distribution events (ex:
[S(n)[ > 3.0), which are moreover often precisely the event of interest. Fortunately we have a
solution to this problem.
3.7 Bahadur-Rao
We want here to study specically the tail distribution of N with events on the formP(N n)
with large n (or P(N n) with small n). For all t > 0 let us rst notice that we can use the
Markov inequality to write: P(N n) = P(e
tN
e
tn
) E[e
tN
]/e
tn
= exp((t) tn). By
taking the smallest of these bounds for t > 0 we hence get: logP(N n) () n with
dened by
/
() = n. This upper bound, known as the Chernoffs Bound (CB), is often
surprisingly sharp for events located in the tail distribution. By dealing symmetrically with
P(N n) and t < 0 we hence obtain the following approximation for S(n):
CB(n) =
n
n ()
log(10)
(28)
where
n
= 1 if n E[N], and
n
= +1 if n > E[N].
From the computational point of view, the solution of
/
() = n can be easily determined
numerically using (for example) using the Newton-Raphson sequence (Press et al., 1992).
Starting for a rst guess t
0
(ex: t
0
= 0), one performs t
i+1
= t
i
+ (n
/
(t
i
))/
//
(t
i
) for
i 0 until convergence to . The computation of ,
/
, and
//
being possible thanks to
Lemma 5 and the following formulas:
(t) = G(e
t
)
/
(t) =
e
t
G
/
(e
t
)
G(e
t
)

//
(t) =
e
2t
G
//
(e
t
)
G(e
t
)

e
2t
G
/
(t)
2
G(e
t
)
2
+
e
t
G
/
(e
t
)
G(e
t
)
(29)
with G(e
t
) = [z
0
]u(P +e
t
Q+zQ)
d
v = u(P +e
t
Q)
d
v, G
/
(e
t
) = [z
1
]u(P +e
t
Q+zQ)
d
v,
and G
//
(e
t
) = 2[z
2
]u(P + e
t
Q+ zQ)
d
v.
Moreover, this bound can be further rened using the Bahadur-Rao Theorem (Bahadur & Rao,
1960) and gives the following approximation for S(n):
BR(n) = CB(n) +
n
log 10
_
(1 e
[[
)
_
2
//
()
_
. (30)
From the computational point of view, CB(n) and BR(n) can be computed either with the
power approach with complexities O(log
2
L
3
) in time and O(log
2
L
3
) in memory;
or with the recursion approach with complexities O( L [/[) in time and O(L [/[) in
memory.
On Figure 7 we can see the reliability of the approximations CB(n) and BR(n). Unsurprisingly,
the farther from the center of the distribution, the better are both approximations. We also
observe that BR(n) is a dramatic improvement over CB(n) since it obtains at least two
correct digits of S(n) for all n but on [120, 200]. At the end of previous section, we have
seen that the order 3 NG approximation achieves the same precision for region [80; 240],
hence, by combining both NG
3
(n) (for the center of the distribution) and BR(n) (for the tail
distributions), one can achieve at least two correct digits of S(n) on the whole bulk of the
distribution for a modest computational cost.
4. Discussion
Obtaining the distribution of motif count in random sequences is a very challenging problem
that has attracted considerable attention from mathematicians and computer scientists in the
last fty years. Recently however, a signicant advance has been obtained by connecting
the well-known theory of pattern matching and automata to the Markov chain embedding
technique Lladser (2007); Nuel (2008a); Nuel & Prum (2007). Thanks to this nding, it is now
possible to deal with simple (runs of 1 in binary sequences, single words, etc.) or complex
motifs (PROSITE signature, gapped motifs, etc.) using the same general framework.
Using exact approaches, it is possible to obtain efciently the rst moments of any motif count
N, and even the complete distribution of N. As a consequence, the computation of S(n) is
now tractable for a wide range of motif problems including large datasets or complex motifs.
However, the case of complex frequent motifs in large datasets remains an open problem
(Nuel & Dumas, 2010).
As an alternative to exact computations, a wide range of approximations have been
developed (see Lothaire, 2005; Nuel, 2006b; Reignier, 2000, for a review). We can basically
classify these approximations in three categories: 1) Gaussian approximations (Cowan, 1991;
Kleffe & Borodovski, 1997; Nuel, 2010; Pevzner et al., 1989; Prum et al., 1995); 2) Poisson
approximations Erhardsson (2000); Geske et al. (1995); Godbole (1991); Reinert & Schbath
(1999); Roquain & Schbath (2007); 3) large deviations approximations Denise et al. (2001);
Nuel (2004).
40 20 0 20 40
0
2
4
6
8
S(n)
l
o
g
1
0
(
r
e
l
a
t
i
v
e

e
r
r
o
r
)
Gaussian
NearGaussian
Poisson
Cramer
BahadurRao
(a) G(G[C)G, frequent motif
0 20 40 60
1
0
1
2
3
4
5
S(n)
l
o
g
1
0
(
r
e
l
a
t
i
v
e

e
r
r
o
r
)
Gaussian
NearGaussian
Poisson
Cramer
BahadurRao
(b) A(A[T)A, rare motif
Fig. 8. Relative error in log-scale for various approximations of S(n) (n = 0, . . . , 200) in a
sequence X
1:
(C) = (G) = 0.40.
In this chapter we deliberately left aside the Poisson-based approximations and considered
only two of these approximations: the (Near-) Gaussian approximations with NG
h
(n), and the
large deviations based approximations with CB(n) and BR(n). The reason why Poisson-based
approximations are not considered here is basically practical, these approximations cannot be
directly derived from the formalism of this manuscript and require the introduction of many
tedious notions like clumps, overlapping words and so on. However, we compare here the
performance of all these approximations (including compound Poisson approximations) in
the case where X
1:
(C) = (G) = 0.40 i.i.d. DNA sequence, and for two motifs: the frequent G(G[C)G, and the
rare A(A[T)A.
We can see on Figure 8 the relative error (in log-scale) for all approximations. For Gaussian
approximations, performances are only good in the very center of the distribution (for n very
close to E(n)) for the frequent motif G(G[C)G, and performances are poor almost everywhere
for the rare motif T(A[T)T. This observation to consistent with the well known claim that
Gaussian approximations a more suitable for frequent motif (Lothaire, 2005). It has however
to be pointed out that even in the most favorable case (with highly frequent motif), Gaussian
approximations totally fail to capture the tail distribution of N and hence not suitable for the
highly signicant observations we usually encounter in biological sequences (Nuel, 2006b).
If we consider now the near-Gaussian approximation, taking into account more moments
of N dramatically improve the result for both motifs, but the failure to deal with extreme
distribution events remains.
Compound Poisson approximations are known to be extremely sensitive to the relative
abundance of the motif of interest in the sequence, being more accurate for rare motifs
(Lothaire, 2005; Roquain & Schbath, 2007). It is hence not a surprise to see that Poisson
approximations are totally unreliable for the frequent motif G(G[C)G. For the rare motif T(A[T)T
we naturally obtain much better results but like for Gaussian approximations, and even in
this favorable case, reliability decreases in the tail distribution. Considering that Poisson
approximations are not easily generalizable to motifs dened by regular expressions, that their
computations could be complicated and time consuming, and that their reliability is highly
questionable in some congurations, it seems advisable to avoid their use is most cases.
With large deviations based approximations, we unsurprisingly get a low reliability in the
center of the distribution, but a high reliability in the tail distribution. With Bahadur-Rao
precise approximations, the improvement over the classical Chernoffs bound is quite
impressive, and the complementarity with Near-Gaussian approximations clearly shows
that a combination of both approaches could be a very efcient way to obtain reliable
approximations of S(n) for all n.
In this chapter we gave all the necessary ingredients to assess the signicance score of motif
in a biological sequence using state of the art results, including several unpublished ones:
Lemma 5 which is an extension of the results of Nuel (2010), and the complete Bahadur-Rao
Section which provides interesting improvements over previous large deviations work
(Denise et al., 2001; Nuel, 2004).
Let us nally point out that for the sake of compactness, we have left aside some interesting
questions and extensions like: approximate matching Hopcroft et al. (2001), renewal
occurrences (Nuel, 2006b; Roquain & Schbath, 2007), joint distributions (Nuel, 2008b; Stefanov
& Szpankowski, 2007), dataset with many sequences (Nuel et al., 2010), and sensitivity to
parameter estimation (Nuel, 2006c). Even if some results are already available for these
problems, many questions still have to be answered in the exciting and challenging eld of
the distribution of motifs in random sequences.
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10
A Systematic and Thorough Search for Domains
of the Scavenger Receptor Cysteine-Rich
Group-B Family in the Human Genome
Alexandre M. Carmo
1
and Vattipally B. Sreenu
2

1
IBMC Instituto de Biologia Molecular e Celular and ICBAS Instituto de Cincias
Biomdicas de Abel Salazar, Universidade do Porto
2
The Weatherall Institute of Molecular Medicine, University of Oxford
1
Portugal
2
UK
1. Introduction
The biological function of proteins is largely determined by their individual component
domains, which are segments within the protein sequence that are self-contained and
spatially arranged. These can be catalytic or structural, and define a number of different
features of proteins such as their enzymatic activity, interactions with other proteins, sugars
or lipids, and determine the cellular localization of the proteins that contain them. A number
of intracellular three-dimensionally-arranged domains, such as Src-homology (SH) or
Pleckstrin-homology (PH) domains, define the nature of protein interactions with other
components of the cell, and enable them to interact with their substrates or binding partners.
The specificity of interactions that is given by the domain is unique to its protein. Similarly,
the extracellular part of most membrane-bound or secreted proteins of eukaryotic cells is
also organized in semi-autonomously-arranged blocks that potentially confer multiple
diverse functions to a particular protein. These domains have been classified and grouped
into protein superfamilies depending on the similarity they have with domains of
prototypical proteins, for example immunoglobulin, fibronectin or C-type lectin domains.
Members of these groups are believed to be homologous and to have arisen by divergent
evolution from a common ancestor. Many membrane-bound or extracellular proteins are
comprised of several domains of the same type, but it is not uncommon to find mosaic
proteins containing domains from different superfamilies.
The scavenger receptor cysteine-rich (SRCR) superfamily comprises a group of proteins that
contain one or multiple domains structurally similar to the membrane distal domain of the
type I scavenger receptor expressed by human macrophages (Freeman et al., 1990). Proteins
classified as belonging to this superfamily may contain other types of domains additionally
to the dominant SRCR modules, such as EGF, CUB, LCCL, or other domains. In mammals,
SRCR proteins are typically expressed in cells of the immune system (Resnick et al., 1994),
although some members can be also expressed in non-immune cells and organs, including
liver, kidney, placenta, stomach, brain and heart (Sarrias et al., 2004). Group A domain-
containing SRCR proteins are present in phyla from the most primitive metazoan to


196
vertebrates, whereas group B domain containing SRCR proteins are only found in
vertebrates. Intriguingly, although SRCR proteins can include other domains, no proteins
have been reported to contain group A and B domains simultaneously.
In mammalian species, SRCR group B orthologs are usually very well conserved and
regarding some of the proteins, a high level of conservation is extended to birds and fish.
However, in some cases a human SRCR protein apparently has no corresponding ortholog
in some mammals, and conversely, there are examples of SRCR group B proteins that are
well characterized in a few mammalian species, that have not been described in humans. By
analyzing the human genome, we can now identify all the remaining, still undescribed
genes encoding SRCR group B domains, which will allow us to perform phylogenetic
analysis of the complete set of group B domains. By comprehensive and systematic whole
genome analysis we have found two new putative transcriptional units containing clusters
of potential SRCR domains, and additionally a further putative gene that contains a single
domain. After our thorough search, we are now confident that all proteins containing group
B SRCR domains in the human genome have been identified.
2. The scavenger receptor cysteine-rich group B family
2.1 Biological function of SRCR group B proteins
The cell surface antigens CD5 and CD6, which function in T lymphocytes, are probably the
most well characterized of the family, each containing three extracellular SRCR domains
(Aruffo et al., 1991; Jones et al., 1986). CD5 and CD6 co-associate with each other at the
surface of T cells (Castro et al., 2003; Gimferrer et al., 2003), and are involved in the
regulation of T cell receptor-mediated activation. The extensive characterization of the
interaction of CD6 with its ligand CD166, expressed by antigen presenting cells (Aruffo et
al., 1997), and the identification of different binding partners for CD5 (Biancone et al., 1996;
Calvo et al., 1999; Pospisil et al., 2000; Van de Velde et al., 1991), had initially suggested that
SRCR group B domains participate in intercellular contacts via protein-protein interactions.
Also, the three SRCR domain-containing soluble protein Sp (Gebe et al., 1997) has been
reported to bind to cells of myeloid and lymphoid origin. Also known as AIM (apoptosis
inhibitor expressed by macrophages), API6 (apoptosis inhibitor 6) or CD5L (CD5-like
molecule), Sp is best known for promoting macrophage survival. Therefore, this sub-group
of small SRCR-containing proteins may be described as having a role in cellular
communication, differentiation and activation. However, for most of the remaining
members of the family no such clear function has been established. In particular the lack of
cellular ligands for most of these proteins raises the possibility that a totally different
function for SRCR domains may exist, if indeed SRCR domain proteins share any common
function.
In addition to CD5, CD6 and Sp, the group B SRCR family presently contains five other
proteins, of which two, CD163 and M160, are membrane bound and expressed by
macrophages. CD163 (Law et al., 1993) and M160 (CD163L1) (Gronlund et al., 2000), which
were both identified in human monocytes, are considered a subgroup of the SRCR group B
molecules. No definitive function has been established for these molecules, although CD163
has been described as binding to, and internalizing, tumor necrosis factor-like weak inducer
of apoptosis (TWEAK), thus having a potential role in atherosclerosis (Moreno et al., 2009).
Additionally, CD163 has a detoxifying role in iron metabolism, where by binding to
hemoglobin-complexed haptoglobin it is able to remove hemoglobin from the plasma
of the Scavenger Receptor Cysteine-Rich Group-B Family in the Human Genome

197
(Graversen et al., 2002; Kristiansen et al., 2001). The remaining three members of the group B
SRCR family are secreted glycoproteins of different sizes and structural complexity. DMBT1,
which was identified on the basis of its deletion in a medulloblastoma cell line, is the largest
member of the family, comprising 14 SRCR domains separated by SRCR-interspacing
domains (Mollenhauer et al., 1997). Apart from being secreted, DMBT1 is also found in
association with the plasma membrane of macrophages, although it is not clear whether
there is a specific receptor or the poorly characterized DMBT1 gene may encode a
transmembrane sequence. Once in the membrane, DMBT1 is a ligand for Surfactant protein
D (SP-D), a C-type lectin that binds to exposed carbohydrates (Holmskov et al., 1999). The
SRCR soluble proteins S4D-SRCRB and SSc5D have four and five group B domains,
respectively, and little is known of their functional or binding properties (Gonalves et al.,
2009; Padilla et al., 2002).
However, it has been recently suggested that Sp (Sarrias et al., 2005), DMBT1 (Bikker et al.,
2002), CD163 (Fabriek et al., 2009), CD5 (Vera et al., 2009) and CD6 (Sarrias et al., 2007) are
capable of detecting microbe-associated molecular patterns, and could bind and clear
bacteria or fungi, reaffirming a scavenger-like role for this group of molecules. These
developments notwithstanding, SRCR superfamily proteins may prove to have very diverse
functions, to the extent that the structural properties of the highly conserved SRCR domains
may be the only unifying feature of the family.
2.2 Structure and organization of SRCR domains
Typically, the 100-110 amino acid-long SRCR domains possess a characteristic pattern of
cysteine residues that establish intra-domain disulfide bridges and contribute to the overall
architecture of the compact domain. The number of cysteine residues and their distribution,
together with the organization of the genomic sequence encoding each domain, divide the
SRCR family into two groups, A and B. Group A domains are encoded by split exons, and
typically have six cysteine residues establishing three disulfide bonds. Group B domains, on
the other hand, are encoded by a single exon and have eight cysteine residues, whose
distribution is remarkably conserved in nearly all known domains (Fig. 1).
So far, eight human SRCR group B proteins have been described, Sp, CD5, CD6, S4D,
SSc5D, CD163, M160 and DMBT1 that contain three to fourteen SRCR domains. Their
encoding genes are dispersed throughout the genome, however a few highly similar pairs
such as CD5-CD6 and CD163-M160 are located on the same chromosome. The identity
between individual domains of different SRCR group B proteins varies from 20 to 80%, and
phylogenetic analysis suggests that they have evolved by sequential intragene duplication,
although there are examples that suggest they may have evolved in some cases by inter-
protein domain shuffling. Only four SRCR domains have been characterized by X-ray
crystallography, and of these three are group A SRCR domains, those of hepsin, a cell
surface serine protease involved in cell growth and maintenance of cellular morphology
(Somoza et al., 2003), M2bp, a tumor associated antigen and matrix protein (Hohenester et
al., 1999), and MARCO, a trimeric SRCR group A protein expressed by macrophages and
dendritic cells that recognizes polyanionic particles and pathogens (Ojala et al., 2007). The
crystal structure of the membrane proximal domain of CD5 (Rodamilans et al., 2007),
together with an NMR solution structure of domain 1 of CD5 (Garza-Garcia et al., 2008)
constitute the only sources of structural information of SRCR group B domains. Comparing
the structures, it is however apparent that the 3D assembly of the different domains in the
two groups is overall conserved, all displaying a very similar fold.


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Fig. 1. Sequence alignment of domains from group B SRCR superfamily members.
SRCR domains are typically sequences of 100-110 amino acids in length compacted into a
heart-shaped fold, where a six/seven-stranded -sheet cradles an -helix. Strands 1, 3 and
4, together with 7, form a curved sheet that wraps around the core 1 helix. From 4
onwards, the structures start to diverge. It is the sequence of amino acids between the
beginning of the domain and the 4 strand that is best conserved between group A and
group B domains, and that roughly corresponds in the group A proteins to the first of two
exons that encode a full SRCR A domain, and in group B proteins to the first 50 amino acids
of the domain.

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2.3 Homology between SRCR domains
The level of amino acid identity among human SRCR group B domains from different
molecules varies from 20% to 80%, but within the same molecule this level can be higher
and even be identical in some domains (e.g. domains 3 and 7, and 10 and 11 of DMBT1).
Similarly, some molecules are remarkably conserved between species, especially among
mammals, although it appears that some level of conservation can be extended to birds, fish
and amphibians in a few specific cases. There are good indications for there being orthologs
of CD6 in the genomes of T. guttata and D. rerio, and some other examples. Nevertheless, the
structure of SRCR group B-containing molecules is best preserved in mammalian species.
The strong homology of SSc5D domains dates back to the divergence of egg- and non-egg-
laying mammals, while CD163 has clearly conserved orthologs in all mammals, including
non-placental species (Table 1).

Table 1. Homology between human and other mammalian CD163 domains. Numbers
represent percentage of identity between each domain, compared to the human sequence.
The significantly conserved homology of some SRCR orthologs is suggestive of profound
functional constraints acting on these proteins. On the other hand, it appears that not all
human SRCR B group molecules have described orthologs in all mammalian species, and
conversely, that there are some SRCR proteins described in different animals that have not
been reported in man.
Noticeably, bovine WC1 (Wijngaard et al., 1992) does not have a human counterpart, nor do
the mouse SCART molecules (Kisielow et al., 2008). Similarly, the human macrophage
specific receptor M160 is not found in all mammalian species, while the closely related
molecule CD163, also specific to the monocytic/macrophage lineage, is clearly present in all
genomes that we have examined. We have compared the similarity between individual
domains of known and characterized members of the SRCR B group, and the corresponding
domains in the bovine proteins (Table 2). While proteins such as S4D, SSc5D and CD163
show high levels of identity between human and bovine sequences, others like CD5 and Sp
are more distantly related. M160 does not have a straightforward ortholog in cattle, so the
bovine sequence used was of the related molecule M160-like, that is related in turn to the
SCART 1 and 2 molecules present in the mouse.


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Table 2. Similarity between human and bovine corresponding SRCR domains. Percentage
identity between each domain is indicated.

Fig. 2. Relationships between human and bovine SRCR molecules.
There are three groups of genes in the CD163 family: CD163 itself (CD163a), present in all
mammals; M160 (CD163b), so far only found in the genomes of primates and in horses; and

201
SCART (CD163c), of which there are two genes found in the mouse, and the bovine gene
M160-like (Herzig et al., 2010). These sets of genes are related to WC1 genes expressed in
cattle, sheep and swine. To obtain a better idea of the relationship between these families of
genes, we aligned the full sequences of known human and bovine proteins containing SRCR
group B domains using ClustalW and drew the corresponding phylogram (Fig. 2). As can be
seen, there are no direct links between human M160, bovine M160L and bovine WC1,
raising the possibility that either some genes were lost during mammalian evolution, or that
the complete characterization and annotation of the genomes has still not been fully
achieved. Clearly, either hypothesis does not exclude the other.
3. A systematic and thorough search for SRCR domains in the genome
Our hope is that the evolution and function of SRCR domains would emerge when all
members of this protein family have been identified. The advent of the human genome
sequence has allowed us to screen, using bioinformatics-based approaches, for new SRCR
proteins still not described or characterized. We decided to focus on group B molecules,
given that proteins of this type are more conserved, restricted in number, and their
specialized function, in this case immune-related, seems better defined. We performed
searches for new members of the SRCR-SF in the completed human genome sequence by
interrogating the genome using TBLASTN 2.2.20+ (Altschul et al., 1997;
http://www.ncbi.nlm.nih.gov/BLAST). Initially, we screened for new sequences exhibiting
similarity with any or all of the SRCR domains comprising the then known SRCR
superfamily proteins (Gonalves et al., 2009). We expected that, for a given TBLASTN run,
bona fide new SRCR domains would have smaller E values than the best matches of the
search sequence with Group A SRCR domains. According to this criterion, the search
identified the sequences encoding domains within already known and characterized
proteins i.e. CD5, CD6, Sp, S4D, CD163, M160 and DMBT1. Additionally, we identified a
cluster of five new SRCR domains, which we further investigated and that later resulted in
the cloning and characterization of SSc5D, a molecule secreted by macrophages and that
comprises five SRCR group B domains (Gonalves et al., 2009).
A caveat in our methodology was that not all group B domains were identified using this
strategy. The most divergent domains, namely those of CD5, were not retrieved in all
searches, and in particular CD5-d1 was rarely identified as having a clear homology with
any other group B domain. Sequence alignment of all group B domains (Fig. 1) highlights
the striking differences of CD5 sequences, and also to some extent of the CD6 domains,
when compared with other sequences that are remarkably similar to each other. In order
to perform a more rigorous search for all putative SRCR group B domains, we conducted
a comprehensive systematic search using PSI-BLAST (Altschul et al., 1997) to find distant
homologs. All known SRCR domains were used as queries to search iteratively against
human non-redundant database with the target sequence length set to 250. BLOSUM62
amino acid substitution matrix with gap open penalty 11, and extension penalty 1, was
used. Sequence masking was disabled and the PSI-BLAST threshold was set to 0.005.
While searching, each PSI-BLAST query was iterated including new hits from the
previous search until it converged, i.e. no new hits were found in subsequent searches.
After each search iteration, results were checked for new SRCR proteins. This meticulous
and robust method picked up all known SRCR domain-containing proteins along with
novel proteins (Table 3).


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Protein Number of SRCR Domains Chromosome
Sp 3 3
S4D 4 7
DMBT1 17* 10
CD5 3 11
CD6 3 11
CD163 9 12
M160 12 12
SSc5D 5 19
8D 8 10
D11
#
11 10
HHIPL1 1 14
Table 3. List of SRCR-containing proteins. * - DMBT1 has been described as containing 14
SRCR domains; # - annotated as a pseudogene; in red denotes new SRCR domains from
uncharacterized proteins.
From this genome-wide search we obtained a total of 76 SRCR group B domains distributed
in 11 genes, each putatively encoding a varying number of SRCR domains. The eleven genes
are spread across the genome on seven different chromosomes: chromosome 10 contains
three SRCR group B-encoding genes, chromosomes 11 and 12 contain two each, and
chromosomes 1, 7, 14 and 19 each contain a copy of just one SRCR-encoding gene. Among
these genes and in addition to known domains from characterized genes, our search has
uncovered 23 new putative group B domains, three of which represent previously
unreported domains localized within the DMBT1 gene. The DMBT1 gene thus putatively
encodes a maximum of 17 SRCR domains. Some controversy has existed on the number of
SRCR domains within the DMBT1 molecule. Like other SRCR-containing proteins (Castro et
al., 2007; Padilla et al., 2002), DMBT1 can be expressed as different isoforms arising by
alternative splicing, which include or exclude individual SRCR domains (Mollenhauer et al.,
1999). It is possible that the new DMBT1 domains have not been previously reported
because they are not expressed in the tissues or cells investigated, however it is also
plausible that the exons coding for these domains have been silenced during evolution and
are now non-functional.
The remaining new domains belong to 3 new putative genes, one 8 domain-encoding gene
(8D), one gene, annotated as a DMBT1-like pseudo-gene, that encodes 11 fragments of SRCR
domains of variable lengths (D11), and a gene encoding a putative Hedgehog interacting
protein-like 1 molecule (HHIP-like 1), which contains a single SRCR domain.
In order to analyze the sequence conservation and diversity of SRCR domains, we aligned
all individual 76 SRCR group B domains using ClustalW2 (Thompson et al., 1994) with the
default substitution matrix (Gonnet series) and gap opening and extension penalties of 10
and 0.2 respectively. Due to the sequence diversity in SRCR domains, several insertions and
deletions were found in the multiple sequence alignment (Fig. 3).
To locate sequence patterns as well as conserved amino acids, the multiple sequence
alignment was used to create a WebLogo (Crooks et al., 2004; http://weblogo.berkeley.edu).

203

Fig. 3. Multiple sequence alignment of all SRCR domains.
The overall height of the stack indicates the sequence conservation at that position, while the
height of symbols within the stack indicates the relative frequency of each amino acid at that
position. It is apparent from the WebLogo that, although sequences vary substantially
between SRCR domains, all cysteine residues (colored in red) are conserved across the
family (Fig. 4).

Fig. 4. Multiple sequence alignment of SRCR domains in WebLogo format.


204
In order to estimate evolutionary relationships among SRCR domains, we performed a
phylogenetic analysis utilizing the maximum likelihood phylogenetic reconstruction
method (proml) available in the phylip phylogenetic package (Felsenstein, 1989). Gaps were
trimmed from the SRCR multiple sequence alignment prior to tree building and the Jones-
Taylor-Thornton probability model employed with constant rate of change among sites. The
reliability of internal branches was subsequently evaluated using 100 bootstrap samplings.
SRCR domains exhibit very complex evolutionary relationships (Fig. 5). In the reconstructed
phylogenetic tree, intra-protein domain clustering as well as inter-protein domain clusters
were observed. Intra-domain clustering, as in the case of DMBT1, strongly suggests the
evolution of these domains via sequential intragenic duplication. At the same time it is
difficult to understand the inter-protein domain similarities. CD5, CD6, Sp, M160 and
CD163 exhibit more diverse relationships. Among them, SRCR domains show greater inter
than intra protein similarities. Given their low sequence similarities, it is uncertain whether
the domains evolved through gene duplication and accumulated mutations have reduced
sequence similarity, or if it is through a convergent evolution mechanism subsequent to
domain shuffling. The similarities of domain pairs M160_d4-CD163_d1, M160_d7-CD163_d4
M160_d8-CD163_d5, M160_d9-CD163_d6, M160_d10-CD163_d7, M160_d11-CD163_d8,
M160_d12CD163_d9, CD5_d1-CD6_d3 and CD5_d2-S4D_d4 are strongly suggestive of
inter-protein domain shuffling.
4. Concluding remarks - the completion of the SRCR group B family
In contrast to the complexity and variety of large protein families such as the G protein
coupled receptor (GPCR) superfamily, which has nearly 800 genes in the human genome,
corresponding to roughly 4% of the full protein-encoding genome, group B of the scavenger
receptor cysteine-rich superfamily appears to be much more limited. So far it includes only 8
members in the entire human genome, although there are additionally 3 proteins described
in other mammalians; SCART1 and SCART2 initially found in mice, and the 11 SRCR
domain-containing protein WC1 expressed in cattle, sheep and swine. Also, the function of
mammalian SRCR proteins seems to be restricted to the immune system, although the exact
nature or biological role of the family is still to be fully determined.
In this study we set out to identify the remaining members of the SRCR group B family in
order to obtain a clear understanding of the biological significance of this important group
of proteins and to clarify some as yet unresolved questions regarding their evolution in
mammalian species. We searched the human genome for the presence of SRCR-encoding
genes using as probes the amino acid sequence of all reported human SRCR domains.
Interestingly, one of the new members we have identified, HHIP-like 1, contains a single
SRCR domain, which is unknown in the family. Moreover, the amino acid sequence
corresponding to the SRCR domain constitutes only a small fraction of the total of the
putative protein (13%). This is in contrast with most other members, whose amino acid
content corresponding to SRCR domains relative to the whole of the protein is significantly
higher, varying between 32% (SSc5D) and 92% (Sp). HHIP-like 1 is related to Hedgehog
interacting protein, a regulatory component of the Hedgehog signaling pathway (Chuang
and McMahon, 1999). However, unlike HHIP-like 1, HHIP does not contain an SRCR
domain.

205

Fig. 5. Maximum likelihood phylogenetic estimation of human SRCR domains. Internal
branch reliability was assessed using bootstrapping method (100 bootstrap replicates).
Branches observed in more than 75 of 100 bootstrapped re-sampling are shown in red.
The second cluster of SRCR domains we have uncovered is located on chromosome 10 and
includes 8 such domains, thus we provisionally termed it 8D. Using the predicted exon-
derived protein sequence, we BLAST-searched other mammalian genomes and the proteins
that we retrieved which were most similar to human 8D were mouse SCART1, bovine
M160L, and mouse SCART2, whose ClustalW alignment scores were 70, 66 and 57,
respectively (Fig. 6). Human 8D and mouse SCART1 have 64% identity for the entire
sequence, while some individual SRCR domains share identities of close to, and even above
80%. We thus believe that 8D is the human ortholog of mouse SCART1. It remains to be seen
whether human 8D can be expressed and produce a mature and functional protein,
although we have detected several 8D transcripts of different sizes (C Gonalves and A
Carmo, unpublished).


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Fig. 6. Sequence alignment of mouse SCART1, human 8D and bovine M160-like protein.
The last new set of domains we have identified is located in a gene also on chromosome 10,
but has been annotated as a non-coding pseudogene. Analysis of its putative sequence
derived from the exon-like sequences in fact reveal that some stretches of several of the
SRCR domains are missing, adding to a number of frameshifts and premature stop codons.
Curiously, 11 SRCR-like domains can be identified, exactly the same number as the typical
bovine WC1 protein. Comparison between the two sequences has failed however to
definitely determine whether these two genes have the same evolutionary origin, as
individually identifiable SRCR or SRCR-type domains seem to have already drifted apart
significantly.
With the recognition of the three new genes, albeit none of them proven to be functional as
yet together with the detection of three new putative SRCR-encoding sequences present in
the DMBT1 gene, we are confident that we have completed the identification of the full set
of scavenger receptor cysteine-rich group B domains in the human genome.

207
5. Acknowledgements
We thank Dr. Simon Lee for reviewing this manuscript. The work in Alexandre Carmos
laboratory is funded by FEDER through the Programa Operacional Factores de
Competitividade COMPETE, and by FCT Fundao para a Cincia e a Tecnologia.
Vattipally Sreenu is funded by the University of Oxford.
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11
Assessing Multiple Sequence
Alignments Using Visual Tools
Catherine L. Anderson
1
, Cory L. Strope
2
and Etsuko N. Moriyama
2,3

1
Department of Computer Science and Engineering
2
School of Biological Sciences and
3
Center for Plant Science Innovation
University of Nebraska-Lincoln,
U.S.A.
1. Introduction
Bioinformatics and molecular evolutionary analyses most often start with comparing DNA
or amino acid sequences by aligning them. Pairwise alignment, for example, is used to
measure the similarities between a query sequence and each of those in a database in BLAST
similarity search, the most used bioinformatics tool (Altschul et al., 1990; Camacho et al.,
2009). Evolutionary history among sequences can be reflected better when more than two
sequences are aligned, in a multiple sequence alignment (MSA). When building an MSA, we
assume that the sequences compared are derived from a common ancestral sequence. Then
the process of MSA building is to infer homologous positions between the input sequences
and place gaps in the sequences in order to align these homologous positions. These gaps
represent evolutionary events of their own. Gaps (also called indels) are caused by either
insertions or deletions of characters (nucleotides or amino acids) on a particular lineage of
sequences during the evolution. Building an MSA is, therefore, to reconstruct the
evolutionary history of the sequences involved. While it is easy to understand that the
quality of MSAs affects the quality of phylogenetic tree reconstruction, the effect of MSA
quality reaches far beyond this. Some examples of bioinformatics methods that utilize
information extracted from MSAs include: profile building in similarity search (e.g., PSI-
BLAST: Altschul et al., 1997), motif/profile recognition (e.g., PROSITE: Hulo et al., 2008),
profile hidden Markov models for protein families/domains (e.g., Pfam: Finn et al., 2010),
and protein secondary-structure prediction (for review, see Pirovano & Heringa, 2010).
There are numerous bioinformatics and molecular evolutionary analyses that are affected by
MSA quality and they can be benefited by having reliable MSAs.
Despite the significance of having good MSAs, assessing MSA quality is far from
straightforward. Measuring the quality of MSAs requires two components: a benchmark
dataset and a scoring method. A benchmark dataset includes reference alignments. These
alignments are considered to represent the evolutionary history of the sequences truthfully.
The same set of sequences included in a reference alignment is then aligned using the MSA
methods to be tested. The reconstructed MSA can be compared with the reference MSA using
a scoring method and the quality of the reconstructed MSA is assessed compared to the


212
reference MSA. Problems exist both in benchmark MSA datasets as well as in the methods
used to measure the MSA quality.
The majority of benchmark MSA datasets are built on real sequences by aligning structural
elements and in some cases with hand-curation (e.g., PREFAB: Edgar, 2004b; OXBench:
Raghava et al., 2003; HOMSTRAD: Stebbings & Mizuguchi, 2004; BAliBASE: Thompson et
al., 2005; Thompson et al., 2011; SABmark: Van Walle et al., 2005). Since the true evolutionary
history of the sequences included in these datasets is unknown, positional homologies
among sequences are unknown and the accuracy of these reference MSAs is subjective
(some issues on benchmark datasets, see Edgar, 2010). Some other benchmark datasets are
generated by simulating sequence evolution based on specific molecular evolutionary
models (e.g., IRMBASE: Subramanian et al., 2005). The advantage of these simulated datasets
is that the evolutionary history of sequences (the guide tree) is known and the true
alignment is given as an outcome of the simulation. Since the evolutionary history is known,
these datasets can be used to assess the quality of both MSAs as well as phylogenetic
reconstruction methods. The disadvantage is that the biological correctness of the simulation
relies solely on the evolutionary models used.
Issues also exist in the methods used to measure the quality of MSAs. While a number of
statistics has been proposed (e.g., Position Shift Error score: Cline et al., 2002; sum-of-pairs
score and column score: Thompson et al., 1999), there is no definite answer how to measure
'biological correctness' of MSAs. It remains for the end user to incorporate the statistics into
their evaluation of this 'biological correctness'.
Due to its significant impact on many bioinformatics and molecular evolutionary studies,
MSA is one of the most scrutinized bioinformatics fields (Kemena & Notredame, 2009;
Thompson et al., 2011). However, assessment of MSAs is usually reserved for power users.
Often regular users simply run one MSA method and proceed to the next analysis without
examining their alignment output (Morrison, 2009b). Considering how MSA quality affects
the outcomes of further analysis, assessment of MSAs, however, should be included as
regular part of sequence analysis. In order to facilitate comparative analysis of MSAs, we
recently developed a software package called SuiteMSA (Anderson et al., 2011). SuiteMSA
provides several alignment-viewing tools that allow the user to compare MSAs both
visually and quantitatively. SuiteMSA also includes a feature-rich biological sequence
simulator, indel-Seq-Gen v2.1 (Strope et al., 2009), with a user-friendly graphical interface,
allowing the users to generate their own benchmark alignments for testing various MSAs.
In this chapter, we first review some of the statistics used to assess the quality of MSAs
focusing on those used in SuiteMSA. We then describe how MSA comparison can be
actually performed using various MSA viewers available in SuiteMSA. Five examples are
chosen from diverse types of alignment problems: proteins with secondary structures,
transmembrane proteins, proteins with length variation, simulated protein sequences, and
ribosomal DNAs. These comparisons illustrate how various MSA methods perform
differently based on their underlying assumptions. We also discuss how different alignment
statistics should be used for assessing MSAs and their limitations.
1

1
All input files and alignments shown in this chapter are available from the following website:
http://bioinfolab.unl.edu/~canderson/SuiteMSA/supplement.html

Assessing Multiple Sequence Alignments Using Visual Tools

213
2. Statistics used to assess multiple sequence alignments
There are two types of alignment statistics. The first type of statistics is used to characterize
a single alignment for the level of conservation in each alignment position and for various
gap measures. These are descriptive measures for a specific alignment and should not be
interpreted as a measure of the alignment quality. The second type of statistics can be used
to compare any two alignments containing the same sequences.
2.1 Descriptive statistics on a single multiple sequence alignment
We describe the following two descriptive statistics: information content and average
hydrophobicity. Both are calculated on a per column basis.
2.1.1 Information content
The Shannon entropy is a measure of the amount of uncertainty (Shannon, 1948). When it is
applied to MSA analysis, it is interpreted as a measure of the diversity of characters within a
given alignment column (Schneider & Stephens, 1990). The amount of information
conveyed, or information content, is given by the decrease in this uncertainty and represents
the level of sequence conservation within a column.
Formally defined, the entropy for the k
th
column of an alignment is given as:

2
( ) - ( , )log ( , )
s k
H k f s k f s k
e
=

, (1)
where s is any character contained in column k and f(s,k) is the frequency of s as it appears in
column k. If there are x
s
of the character s in the column that has x of non-gap characters,
f(s,k) is calculated as x
s
/x. The information content in the k
th
column is given as:

2
( ) log ( ) I k S H k = , (2)
where S is the number of character types for an alignment (4 for a nucleotide alignment and
20 for an amino acid alignment). Both H(k) and I(k) have their units in bits.
It can be seen from these equations that the higher the number of distinct characters within a
column, the higher the entropy value (H) and thus, the lower the information content (I) in
the column. For a completely conserved column c, one which contains only one type of
characters, the entropy H(c) is 0; thus it contains the maximum amount of information. For a
nucleotide alignment this maximum value is 2, while for an amino acid alignment it is 4.32.
Note that gaps are not considered in calculating f(s,k) in equation (1). Excluding gaps from
calculation could inflate the information content for a column that contains many gaps. A
single character in a column of gaps, for example, can be erroneously attributed a maximum
information content. In order to compensate for this situation, the column information
calculation is normalized by multiplying each columns information content by the
proportion of non-gap characters present in the column (Schneider & Stephens, 1990).
While the information content is a measure applicable to a single alignment, it can be useful
to compare the information statistics among alternate alignments for trends.
2.1.2 Average hydrophobicity
Hydrophobicity is one of the most useful properties of amino acid residues, which is
directly related to the function and structure of proteins. Many different types of


214
hydrophobicity indices are available (Kawashima et al., 2008). By plotting hydrophobicity
values along the sequence, the presence of functional/structural regions (e.g., membrane-
spanning regions in transmembrane proteins or core regions in globular proteins) can be
predicted. For MSA analysis, comparing the distribution of hydrophobicity along the
alignment among different MSAs can provide a visual aid for evaluating the consistency
between alignments. Equation (3) below shows how the average hydrophobicity for column
k, h(k), is calculated for an alignment containing N sequences:

1
( )
N
i
i
h
h k
N
=
| |
|
\ .
=

, (3)
where h
i
is the hydrophobicity index value of i
th
residue of column k. In SuiteMSA, the
hydrophobicity index provided by Kyte and Doolittle (1982) is used and the value of 0 is
assigned for a gap.
2.2 Measuring the similarity between two multiple sequence alignments
As mentioned earlier, many statistics have been proposed to compare two MSAs. The sum-
of-pairs score (SPS) and the column score (CS) are the two used most often. Both scores were
proposed by Thompson et al. (1999). The values of these two scores react differently to
varying inconsistency between MSAs compared.
When comparing two alignments, one is referred to as the reference alignment and the other
the test alignment. The test alignment is compared against the reference. If the reference
alignment is known to be 'correct', these statistics can be used to measure the alignment
quality. As mentioned before, however, the 'correctness' of an alignment can be highly
subjective in the case of many available benchmark datasets. An alignment can be said to be
truly 'correct' only if its exact evolutionary history is known and if the alignment reflects it
correctly. Usually it is possible only if the alignment was generated by a sequence evolution
simulator. Even if the 'true' alignment can be obtained by sequence simulation, however,
'biological realism' of the evolutionary model used with the simulation becomes an issue. In
this chapter, SPS and CS are thus used more as general comparison measures.
2.2.1 Sum-of-pairs score (SPS)
To calculate the SPS for a test MSA against the reference MSA, each pair of characters within
an alignment column is treated as an alignment unit. The per-column SPS is the number of
alignment units within a specific column of the test alignment that are also aligned in the
same column of the reference alignment. The total of all per-column scores from the entire
alignment is obtained and normalized by dividing by the total number of character pairs.
This is formally defined as follows:
i. Let an alignment of length M containing N sequences be an N by M array, A. Then the
character in the i
th
sequence and k
th
column of the alignment is identified as A
ik
.
ii. Let there be two alignments for comparison: alignment A
r
(referred to as the reference
alignment) of length M
r
containing N

sequences and alignment A (referred to as the test
alignment) of length M containing N sequence, where M
r
and M

can be but are not
required to be equal.
iii. To examine the k
th
column of A, consisting of elements A
1k
, A
2k
, A
nk
, let p
ijk
be defined
as:


215

(4)
iv. Then the score for k
th
column of A is defined as:

. (5)
v. The score for the full alignment A

is given as:

, (6)
where S
rk
is the score for the reference alignment, A
r
. This reference score is calculated
as S
rk
= x(x-1)/2 where x is the number of characters in column k excluding gaps.
The maximum possible SPS is a value of 1.0 when A = A
r.
The SPS is not symmetric in that
the score will be different if the reference and test alignments are switched.
2.2.2 Column score (CS)
To calculate the CS, the test and reference alignments are compared column-wise. The
column score is the number of 'matched' columns between the test alignment and the
reference alignment divided by the total number of 'considered' columns in the test
alignment. This is formally defined as follows:
i. For the k
th
column of A:

(7)

ii. The column score for the full alignment A

is given as:

. (8)
In SuiteMSA, two types of CS are calculated: un-gapped and gapped.
Un-gapped CS: This score considers only un-gapped columns (columns that have no gaps),
where M of equation (8) equals the number of un-gapped columns in the alignment (shown
in red in Fig. 1). For example, if an alignment has 500 columns and only 200 contain no gaps
and of these 200, 150 columns are exactly as they appear in the reference alignment, then the
un-gapped CS is given as 150/200 = 0.75. The disadvantage to these criteria is that very
gappy alignments with very few un-gapped columns can still produce a high column score
if those un-gapped columns are all 'matched'. For instance, a test alignment of any length,
even if only one column is un-gapped and matches a column in the reference alignment, will
yield a column score of 1.0.
p
ijk
= 1 if A
ik
and A
jk
of alignment A are in the same column of A
r
,
p
ijk
= 0 otherwise.
S
k
= p
ijk
j=i+1
N
i=1
N
SPS =
S
k
k=1
M
|
\
|
.
|
|
S
rk
k=1
M
r
|
\
|
.
|
|
C
k
= 1 if all the characters in the column k of alignment A are matched in alignment A
r
,
C
k
= 0 otherwise.
CS =
C
k
k=1
M
|
\
|
.
|
|
M
k


216

Fig. 1. Illustration of the column score calculation. In the Test alignment, 'un-gapped'
columns are shown in red. 'Un-gapped matched' columns are indicated with red '+' under
the alignment. For 'gapped' CS, all but 5
th
column of the Test alignment are considered and
these columns are shown in blue as well as red. However, only those columns indicated
with '+' (both red and blue) are counted as 'matched' against the Reference alignment. In this
example, 'un-gapped' CS is 0.5 (2 out of 4 columns are matched) and 'gapped' CS is 0.4 (4 out
of 10 columns are considered to be matched).
Gapped CS: This score considers columns that contain more than 20% non-gap characters. To
be 'matched' the characters that appear in a column of the test alignment must appear in a
column of the reference alignment with no additional characters. For example, in Fig. 1, all
but 5
th
column of the Test alignment are considered. The columns 6-11 are not counted as
'matched'. This is because, for example, while in the Test alignment, 'G' of T1 position 9 is
aligned only with 'G' of T6 and T7, in the Reference alignment, 'G' of T1 position 9 is aligned
with 'G' of T2 as well as T6 and T7. The advantage to 'gapped' CS is that it allows more
columns to be considered; columns with gaps can be matched if the same non-gap
characters (but no other characters) are aligned in the reference alignment. This does offset
the disadvantage of the potentially inflated un-gapped CS mentioned before.
Exclusion of any alignment columns that include gaps can be justified since gaps represent
evolutionary events that are often not traceable. They are either the insertion of new
characters, the deletion of existing characters, or a combination of the two. Therefore, while
they are represented by the same gap symbol in the alignment, they are not equivalent. It is
often not possible to infer if a gap in one alignment was generated by the same event as a
gap in the second alignment. On the other hand, excluding all alignment positions with gaps
even for those containing only a small number of gaps may not be desirable. In SuiteMSA, as
described above, a column is considered as long as it contains a number of non-gap
characters above the 20% threshold. A third column score is also provided in SuiteMSA as '%
consistency', which considers all columns regardless of the number of gaps. Comparing these
values can help assessing the difference between two alignments.
2.2.3 Implementation of SPS and CS
In addition to SuiteMSA, several implementations of SPS and CS are available as listed in
Table 1. Note that not all of these programs generate the same value for the same alignment.
The difference is caused by different criteria used to define, for example, 'matched' columns
and which columns should be 'considered' for counting. When comparing scores, due to this
inconsistency among programs, it is necessary to use the same implementation of scoring
methods.


217
Program Reference Note
bali_score (Thompson et
al., 1999)
standalone; C program; MSF format.
qscore (Edgar, 2004b) standalone; C++ program; calculates Q score (SPS), TC (CS),
Modeler score, and Shift scores; fasta format.
VerAlign available from http://www.ibi.vu.nl/programs/veralignwww
MSF format.
SuiteMSA (Anderson et
al., 2011)
part of the GUI software; fasta format.
Table 1. Programs available to calculate SPS and CS. The actual SPS and CS values for
alignments discussed in this chapter given by different programs are available from our
website (see footnote 1).
3. Visual inspection of MSAs
In the following sections, using various examples, we will show how MSAs can be
compared using SuiteMSA's visual tools and statistics. See Anderson et al. (2011) and
SuiteMSA User's Manual for detailed description of various tools available in SuiteMSA.
Among the numerous MSA methods currently available, we chose seven MSA methods
listed in Table 2 for comparative analysis. We chose these methods based on their general
popularity in various bioinformatics analyses, their availability, and some of their features
useful for aligning particular types of proteins (e.g., transmembrane proteins).

Method
(version)
Reference Description
ClustalW2
(2.1)
(Larkin et al.,
2007)
Progressive alignment; weights sequences based on
branch lengths and adjusts gap penalties; one of the
earliest methods implemented. http://www.clustal.org/
MUSCLE
(3.8.31)
(Edgar, 2004a,
2004b)
Progressive alignment; fast distance estimation using
kmer counting; iterative refinement using tree-dependent
restricted partitioning. http://www.drive5.com/muscle/
MAFFT
(6.843)
(Katoh & Toh,
2008)
Progressive alignment; L-INS-i method is used for
iterative refinement incorporating local pairwise
alignment information in this study.
http://mafft.cbrc.jp/alignment/software/
Probalign
(1.4)
(Roshan &
Livesay, 2006)
Uses partition function posterior probability estimates to
compute maximum expected accuracy alignments.
[eProbalign] http://probalign.njit.edu/probalign/login
PRANK
(web version)
(Lytynoja &
Goldman, 2005,
2008)
Phylogeny-aware gap handling; not meant for divergent
sequences; recognizes insertions and deletions as distinct
evolutionary events.
[webPRANK] http://www.ebi.ac.uk/goldman-srv/
webprank/


218
Method
(version)
Reference Description
PRALINE
(web version)
(Pirovano et al.,
2008)
Progressive alignment with profile pre-processing;
incorporates secondary structure and transmembrane
information; PSIPRED and Phobious (for GPCR
alignment) chosen for this study.
http://www.ibi.vu.nl/programs/pralinewww/
PROMALS
(web version)
(Pei & Grishin,
2007)
Progressive alignment enhanced with profiles and
secondary structure information; a hidden Markov model
using a combined scoring of amino acids and secondary
structures. http://prodata.swmed.edu/promals/
Table 2. The seven MSA methods compared in this study. All methods are used with the
default options unless noted otherwise.
3.1 Examining a protein MSA with secondary structure prediction
When protein sequences are aligned, it is useful to identify the location of their functional or
structural landmarks to determine if such landmarks are aligned properly. Useful
landmarks include secondary structures, transmembrane regions, and conserved domains
or motifs. Color-coding MSAs based on properties of amino acids also helps determine if the
distribution of different types of amino acids is consistent or varied among sequences.
3.1.1 Inspecting a single MSA
In Fig. 2, eight protein sequences of the lipocalin family (Pfam PF00061; Finn et al., 2010) are
aligned. The lipocalin family proteins are highly divergent at the sequence level yet highly
conserved at the structure level (Flower et al., 2000). The common structural feature among
these proteins is a single eight-stranded antiparallel beta-barrel. The MSA shown in Fig. 2
was originally produced using PROMALS3D (Pei et al., 2008) with manual adjustment
(Strope et al., 2009). Using SuiteMSA's secondary structure viewer, we aligned the lipocalin
MSA with the secondary structures predicted from the eight sequences using PSIPRED
(Jones, 1999). It can be seen in Fig. 2 that eight beta-strand regions (shown as brown-colored
clusters of 'E' letters) are clearly well aligned with very few gaps.
Fig. 2 also shows the per-column information content displayed as a blue bar chart below
the MSA. The information content reflects the level of conservation for each column. This
display is especially useful when dealing with alignments containing a large number of
sequences and/or long sequences. When comparing such large alignments, the information
content display can be used to quickly scan along the alignment to search for, e.g., high
conservation areas (indicated as high information content regions). In Fig. 2, fully conserved
columns (positions 51, 53, 148, 150, and 179 are readily identifiable by the full-height bars. In
fact, these positions are part of the three conserved motifs shared among lipocalin proteins.
These motifs (indicated as M1, M2, and M3 in Fig. 2) are described as "structurally
conserved regions" (SCR1, 2, and 3, respectively) by Flower et al. (2000). SCR1 corresponds
to PROSITE lipocalin motif (PS00213; Hulo et al., 2008).
Several summary statistics are given at the top of MSA Viewer window (Fig. 2). The
following statistics are available:


219

Fig. 2. The alignment of eight protein sequences from the lipocalin family. The MSA Viewer
is used to display the MSA aligned with the predicted secondary structures. Black thick
lines marked with M1, M2, and M3 indicate the locations of the three conserved motifs.
Symbols used for the secondary structure prediction are: H (green) for helix, C (cream) for
coil, and E (brown) for beta-strand. The alignment statistics are shown above the MSA. The
column information content is displayed as a blue bar chart at the bottom indicating the
level of conservation for each column.
- % gaps. The number of gap symbols within the alignment divided by the total number
of characters within the alignment (alignment length times number of sequences). This
should not be confused with the number of insertion/deletion events in the alignment
since an individual event can span multiple positions.
- % conserved. The number of completely conserved columns divided by the total number
of columns. A conserved column is defined as an un-gapped column containing a single
type of characters.
- % columns un-gapped. The number of un-gapped columns divided by the total number
of columns.
- The histogram of character count per column. This histogram represents the gappiness
of the MSA using a non-gap character frequency distribution (the inverse of gap
frequency distribution). For the lipocalin MSA, 73% of the columns have no gap (this is
also shown as % columns un-gapped).
3.1.2 Comparing two MSAs
In Fig. 3A, we compared the previously shown lipocalin MSA (listed as 'Reference') with the
MSA generated by ClustalW2 using the MSA Comparator. Under the blue selection bar and
the green range bar, alignment positions are color-coded for the consistency with respect to
the reference MSA. Blue characters illustrate where completely consistent columns are, and
red characters depict those inconsistently aligned. Compared against the reference,
ClustalW2 MSA is more compacted with very few gaps, making the alignment shorter (201
positions compared to 219 in the reference). We further examined the ClustalW2 MSA using
the secondary structure display function of the MSA Viewer. As illustrated in Fig. 3B, the
ClustalW2 MSA does not have the beta-strand regions (shown as brown-colored clusters of
'E' letters) aligned as well as the reference MSA does.
As mentioned earlier, the information content is the indicator of sequence divergence within a
single MSA, and not a direct comparison between two alignments. However, as shown in Fig.
3A, the information content distributions (blue and green bar charts) can be compared
between the alignments. It is especially useful when dealing with large alignments containing


220

Fig. 3. Comparison of the ClustalW2 MSA with the reference alignment of the lipocalin
family. A. The two MSAs are compared using the MSA Comparator (the reference and
ClustalW2 alignments shown at the top and bottom, respectively). The column SPS display
(brown bar chart) is positioned between the two MSAs and is aligned to the ClustalW2
alignment. At the bottom of the column SPS display is the column score (CS) indicator. The
un-gapped CS uses those columns marked with purple squares, and the gapped CS uses
columns marked with both purple and red squares (small and large squares indicate
'considered' and 'matched' columns, respectively). Summary statistics shown above the
reference alignment include: % consistency, SPS, and two types of CS. B. The MSA Viewer is
used to generate the secondary structure representation for the reference and ClustalW2
MSAs (shown at the top and bottom, respectively). Symbols used for the secondary structure
prediction are: H (green) for helix, C (cream) for coil, and E (brown) for beta-strand.
many/long sequences. On the other hand, SPS is the result of a direct comparison between two
MSAs. The per-column SPS (brown bar chart) displayed in Fig. 3A clearly shows where the test
alignment (ClustalW2 in this case) is consistent (and to what degree) with the reference.
3.1.3 Comparing multiple MSAs
In Fig. 4, we compared MSAs produced by four methods against the reference lipocalin
family MSA (MSA 1). Using the Pixel Plot, we can clearly see different patterns among the
MSAs. The magenta-highlighted areas illustrate how the corresponding characters are
aligned (or not) in each MSA. The PRALINE MSA (MSA 2) is fairly consistent compared to
the reference MSA. This is expected since PRALINE uses secondary structure information
when optimizing the alignments. On the other hand, MAFFT, MUSCLE, and ClustalW2
MSAs show a similar displacement of the same sequences, apparent from the ragged edges


221

Fig. 4. Comparison of the lipocalin family reference MSA (MSA 1) with four reconstructed
MSAs (PRALINE, MAFFT, MUSCLE, and ClustalW2). The Pixel Plot is used to show the
alignment patterns with each non-gap character represented with a solid colored pixel and a
gap with a blank pixel. Characters corresponding to those under the blue selection bar for the
reference MSA are highlighted in magenta in all MSAs. The green range bars for MSAs 2-5
show the column ranges where corresponding characters are located.
of the magenta areas. Alignments generated by MAFFT, MUSCLE, and ClustalW2 (MSAs 3-
5) are roughly consistent to each other, but not consistent with the reference and PRALINE
alignments. All four MSA methods tested produced shorter alignments (201-214 positions)
compared to the reference alignment (219 positions). The shortest alignment was obtained
from ClustalW2 (201 positions).
4. Aligning transmembrane protein sequences
In the previous section, we showed that comparing MSAs and secondary structure predictions
help us assess the quality of MSAs. In this section, we will examine alignments of another type
of proteins, transmembrane proteins.
4.1 G-protein coupled receptors
G protein-coupled receptor (GPCR) proteins contain seven transmembrane (TM) regions.
They constitute a large protein superfamily grouped into three major and several minor
classes (Horn et al., 2003; Vroling et al., 2011). Although the TM regions are relatively
constant in length (22~24 amino acids or aa), the lengths of the N-/C-terminal and loop


222
regions are highly varied especially among different classes (Inoue et al., 2004; Wistrand et
al., 2006). GPCR sequences are also highly divergent. These features make aligning GPCR
sequences a challenge. We sampled 25 protein sequences from three major classes of GPCRs
(Classes A, B, and C). The lengths of these GPCR sequences vary from 201 to 972 aa.
4.2 Alignment of GPCR sequences
Fig. 5 shows the alignment of the 25 GPCRs generated by PRALINE (showing only the first
three TM area). Since PRALINE incorporates information from secondary structure, TM
structure, as well as profiles based on PSI-BLAST similarity search (Table 2), it is expected to
perform well in aligning TM regions. In order to confirm this, TM regions were predicted
for each of the 25 GPCR sequences using MEMSAT3 (http://bioinf.cs.ucl.ac.uk/psipred/;
Nugent & Jones, 2009). The predicted TM structural information was then aligned with the
PRALINE MSA. Fig. 5 shows that the predicted TM regions (depicted with 'X' in green
color) are clearly well aligned and visualized as green-colored clusters. The 'hydrophobicity'
color scheme used for the MSA display as well as the average column hydrophobicity plot
also confirm that more hydrophobic amino acids are found in predicted TM regions.

Fig. 5. Alignment of 25 GPCR proteins generated by PRALINE compared with TM structural
predictions. Using the MSA Viewer, the PRALINE MSA is displayed using the 'hydrophobicity'
color scheme showing hydrophobic amino acids more toward red and hydrophilic amino
acids more toward blue. The predicted TM structure corresponding to each sequence of the
MSA is aligned below. The symbols (based on MEMSAT3 prediction) used to show different
TM structural components are as follows: 'X' (green) for the TM region, '+' (light brown) for
the inside loop, 'I' (brown) for the inside helix cap, '=' (cream) for the outside loop, and 'O'
(yellow) for the outside helix cap. The first three TM regions are depicted as three clusters of
green letter X's. At the bottom of the display is the information content for each column
(blue bar) and the average hydrophobicity for each column (black line plot). The average
hydrophobicity (see equation (3)) is based on the index given by Kyte and Doolittle (1982).


223
4.3 Comparison of GPCR MSAs reconstructed by seven methods
We aligned the 25 GPCR protein sequences using seven methods. The seven MSAs
produced were compared using Pixel Plot in Fig. 6. Compared to the terminal or loop
regions, seven TM regions are expected to have fewer gaps. Using the Pixel Plot we can
confirm such patterns. Approximate areas predicted to have TM regions can be located as
clusters of solid colored pixels. In Fig. 7, the seven MSAs are represented in the predicted
TM structures. The area includes the first five TM regions shown as the green-colored
clusters. Both PRALINE and PROMALS utilize information from secondary structure
prediction (also TM prediction for PRALINE) as well as profiles based on PSI-BLAST
similarity search (Table 2). As expected, in the MSAs reconstructed by these two methods,
predicted TM structures are aligned better than other methods. Other methods with the
exception of PRANK also generated MSAs that aligned the area containing the first three
TM regions relatively well. The rest of the sequences were more difficult for alignment.
Probalign had a difficulty in reconstructing also the third TM region. With all MSA
methods, all positions after the third TM region were not well reconstructed in terms of
conservation of TM regions. The difficulty in aligning the second half of the protein
sequences is likely caused by the large length variation found among GPCR classes,
especially in the fourth and fifth loops (between TM4 and TM5, and TM5 and TM6,
respectively) (Wistrand et al., 2006).
In order to gain more insights on the difference among GPCR protein MSAs quantitatively,
we gathered SPS values from all pairwise comparisons among the seven MSAs. Each of the
seven MSAs was used as the reference and other six MSAs were tested against. Fig. 8 clearly
shows that SPS is not symmetric. As expected, PRALINE and PROMALS, both of which
utilize secondary structure and TM prediction information, had very high SPS' when they
are compared to each other (0.546 and 0.543). Interestingly, using PRALINE or PROMALS as
the reference, MAFFT was found to perform very well although MAFFT does not
incorporate secondary structure nor profile information. It should be also noted that SPS' are
among the highest when Probalign was compared to MAFFT (either as the reference or the
test MSA).
The most drastic difference between the row and column averages of SPS' is found in
PRANK. The SPS' obtained when the PRANK MSA was used as the reference (shown in the
PRANK column) are all higher than those obtained when the PRANK MSA was tested
against others (shown in the PRANK row). This can be explained by the gappy nature of the
PRANK MSA (see Figs. 6 and 7). The PRANK MSA tends to have more gaps because of the
underlying design of the method. It attempts to identify distinct insertions and deletions
and tries not to collapse such independent events into the same column. For the same set of
sequences, the reference alignment that has more gaps has a fewer number of character-
pairs available (denominator in equation (6)) when averaging the total SPS, which tends to
generate a higher SPS. Note also that the phylogeny-aware algorithm used with PRANK
cannot perform well when sequences are too diverged (Lytynoja & Goldman, 2008). With
extremely diverged GPCR sequences, PRANK was not expected to perform very well,
which was indicated by constantly low SPS' obtained with PRANK. Although in the absence
of 'true' reference alignment, low SPS values do not necessarily indicate incorrect alignment
but rather inconsistency between the alignments, virtually no TM region was conserved in
the PRANK MSA (Figs. 6 and 7). We will examine more on PRANK in the next section.


224

Fig. 6. Comparison of seven GPCR protein MSAs. The characters corresponding to the second
TM (TM2) regions are highlighted with magenta-colored pixels. It shows that the TM2 region
is reconstructed well in the MSAs by PRALINE (MSA 1) and PROMALS (MSA 2), relatively
well by MAFFT (MSA 3), MUSCLE (MSA 4) and ClustalW2 (MSA 5), and not very well by
Probalign (MSA 6) and PRANK (MSA 7). For the PRALINE MSA, the positions for the seven
TM regions are as follows: 555-572, 595-611, 641-663, 683-703, 738-756, 813-830, and 854-875,
which roughly correspond to solid-colored regions.


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Fig. 7. Seven GPCR protein MSAs represented in TM structures predicted by MEMSAT3. The
areas covering the first five predicted TM regions are shown. The red boxes indicate the areas
containing amino acids predicted for the second TM regions. Wider red boxes in reconstructed
MSAs indicate TM regions with a higher number of gaps (e.g., Probalign and PRANK). The
symbols representing amino acids predicted for different TM structural components are as
follows: 'X' (green) for the TM region, '+' (light brown) for the inside loop, 'I' (brown) for the
inside helix cap, '=' (cream) for the outside loop, and 'O' (yellow) for the outside helix cap.


226
Reference
Test
PRALINE PROMALS Probalign MAFFT MUSCLE ClustalW2 PRANK [Average]
PRALINE (1,077) 0.543 0.563 0.592 0.474 0.381 0.351 0.484
PROMALS 0.546 (1,078) 0.538 0.568 0.500 0.345 0.344 0.474
Probalign 0.477 0.455 (1,111) 0.520 0.457 0.324 0.362 0.433
MAFFT 0.569 0.546 0.590 (1,081) 0.498 0.346 0.357 0.484
MUSCLE 0.462 0.487 0.526 0.505 (1,445) 0.325 0.322 0.438
ClustalW2 0.383 0.346 0.384 0.362 0.335 (1,041) 0.283 0.349
PRANK 0.231 0.227 0.227 0.245 0.218 0.186 (2,419) 0.232
[Average] 0.445 0.434 0.471 0.465 0.414 0.318 0.337
Fig. 8. Pairwise comparison of the sum-of-pairs scores (SPS) between GPCR protein MSAs
reconstructed by the seven methods. The numbers in parentheses are the alignment lengths
(the number of columns in each alignment). The highest score in each comparison is shown
in boldface.
5. A different perspective on gaps
In this section we highlight the alignment method PRANK, which is unique in
emphasizing a different perspective on the evolutionary process producing insertions and
deletions. As shown in the previous section, it tends to produce more gaps in alignments
compared to other methods. We compare the alignment generated by PRANK with four
other methods.
5.1 Viral envelope glycoprotein, gp120
Lytynoja and Goldman (2008) used the viral exterior envelope glycoprotein, gp120, from
human and simian immunodeficiency viruses (HIVs and SIVs, respectively) as an example
to demonstrate how PRANK works. In this section, we used the same set of sequences they
used (the seed alignment of Pfam Family GP120, PF00516, excluding SIVGB, SIVV1, and
SIVG1). The entry of HIVs and SIVs into the host requires the interaction of the viral gp120
with the cell-surface proteins of the host. In order to avoid the host's immune system,
several regions of the gp120 proteins evolve fast. Fig. 9 shows the MSA of gp120 proteins
compared with the predicted secondary structures.
5.2 Gap treatment
The 'gap' within an alignment is a general expression for two very different types of
evolutionary events. It represents either an insertion of one or more characters or the
deletion of one or more. Both types of events are unobservable, and as such it is difficult to
distinguish which event creates a gap in an alignment. For example, the 'gappy' section of
an alignment, such as the V1 section of HIV/SIV gp120 (Fig. 9), can be interpreted either as
the result of a high substitution rate along with frequent independent deletions or as the
result of frequent independent short insertions and deletions. Optimization functions used
in most MSA methods over-infer the former scenario, stacking independent insertions in the
same column and potentially erroneously inflating substitution rates in such regions. Using
phylogenetic information, PRANK, on the other hand, allows for the inference of both
deletions and insertions as separate events.


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Fig. 9. Reference MSA of HIV/SIV gp120 proteins. The reference alignment was based on
the seed alignment of Pfam Family GP120 (PF00516). The secondary structure is predicted
for each sequence by PSIPRED (Jones, 1999) and is displayed using the MSA Viewer. The
symbols depicting different secondary structures are as follows: H (green): alpha-helix, E
(brown): beta-strand, and C (cream): coil. The MSA region shown above contains the N-
terminal conserved area and the V1 variable region (positions 100-150). The predicted
regions of the beta-strands (positions 50-53 and 93-96) and the alpha-helix (positions 66-78)
correspond to the known HIV gp120 protein structure.
Progressive alignment methods such as ClustalW2, MAFFT, and MUSCLE build an
alignment based on aligning the profiles of previously aligned sequences. The presence of a
gap in the profiles is not checked to determine if the addition of another gap is
parsimonious with the guide tree. The decision of whether adding a gap or not is based on
the optimization function score. Since the inference of additional gaps penalizes the
optimization function score, it often results in incorrectly matching potentially independent
insertions, creating incorrect homologies.
PRANK attempts to avoid the above-mentioned pit-falls in progressive alignments by
utilizing "phylogeny-aware" handling of gaps and treating insertions and deletions
differently. The overall effect of the PRANK method compared to other progressive
alignment methods is that the alignment is extended due to the separation of the
independent insertions. As Lytynoja and Goldman (2008) stated, "the resulting alignments
may be fragmented by many gaps and may not be as visually beautiful as the traditional
alignments, but if they represent correct homology, we have to get used to them."
5.3 Comparison of the PRANK MSA with others
We aligned the set of 21 gp120 sequences using PRANK and other five alignment methods.
For PRANK, we reconstructed the phylogeny using PhyML 3.0 (Guindon et al., 2010) and
used it as the input phylogeny (with rooting between HIV1 and HIV2 clusters, the topology
was identical with the one given in Lytynoja & Goldman, 2008). As shown in Fig. 10, the


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Fig. 10. Comparison of gp120 MSAs. The Pixel Plot is used to compare five reconstructed
MSAs (MSA 2: PRANK, MSA 3: PROMALS, MSA 4: Probalign, MSA 5: MAFFT, and MSA 6:
MUSCLE) with the reference alignment (MSA 1, based on the seed alignment of Pfam
Family GP120, PF00516). The area highlighted in magenta color is part of the V1 variable
region, where the patterns show that the PRANK MSA is highly inconsistent with other
MSAs.
major differences among MSAs are found starting at the first highly variable area, V1.
Within this area, PRANK infers far more insertions than the other methods. The number of
sites covered by the blue selection bar in the reference alignment (MSA 1) is 53. The
corresponding sites in the other alignments are spread over from 51 columns with MAFFT
(MSA 5) to 84 columns in PRANK (MSA 2).
Table 3 summarizes alignment statistics. As expected, PRANK generated the longest
alignment. This is indicated in the PRANK MSA having a higher % gaps, lower %
consistency, and lower % no-gap columns. Note also that the reference alignment used was
the Pfam seed alignment, which in principle was generated using an alignment strategy
similar to methods other than PRANK. These comparisons clearly illustrate the point made
by Lytynoja and Goldman (2008). Depending on the MSA method used, a very different
evolutionary mechanism would be emphasized to explain fast evolving gp120 sequences:
either accelerated substitution rates or extremely high rate of short insertions or deletions.
Another important point is that scores devised for MSA comparison (e.g., SPS) should be
used with the knowledge of the assumption underlying the design of the method used as
well as the nature of the reference alignment.


229
Method SPS
CS with
no-gap
CS with
gaps
%
consistency
MSA
length
(aa)
%
conserved
% gaps
% no-gap
columns
Reference - - - - 563 14.90 14.50 71.20
PRANK 0.872 0.845 0.702 53.08 633 13.60 23.90 61.30
PROMALS 0.919 0.855 0.775 68.24 548 15.30 12.10 76.60
Probalign 0.920 0.838 0.761 62.50 579 15.00 16.80 72.70
MAFFT 0.907 0.827 0.727 63.90 557 15.40 13.60 74.70
MUSCLE 0.910 0.926 0.750 66.20 548 15.50 12.10 77.60
Table 3. Alignment statistics for gp120 MSAs. SPS, CS, and % consistency are obtained
against the reference alignment. The highest value in each comparison is shown in boldface.
Fig. 11 illustrates a comparison between the MSAs generated by MAFFT (top) and
PRANK (bottom). In Fig. 11A, the region under the blue selection bar for the MAFFT MSA
(positions 104 to 128; 25 aa long) is more compact than the region covered by the
corresponding amino acids in the PRANK MSA as indicated by the long green range bar
(ranging from positions 106 to 159; 54 aa long). In this region, PRANK shows, for
example, two independent insertions marked with light blue boxes ('GL' and 'MIR') both
happening in SIV/HIV2 sequences. In the MAFFT MSA, these two sequences are part of a
much longer insertion region unique to SIV/HIV2, implying that frequent deletion events
shortened this region in various HIV2. Another insertion found in HIV1 by PRANK,
'SSSLR' (in a light green box), is shown to be almost independent. However, in the
MAFFT MSA, the corresponding region appears to have experienced many deletion
events instead. This shows the "gap magnet" phenomenon found in many progressive-
alignment methods. Fig. 11B from the same MSA area highlights another possible artifact
often found in MSAs generated by progressive alignment methods. In the red area in the
MAFFT MSA, all sequences are aligned (matched) generating the "collapsed insertions",
implying homologous relationships among these sequences. However, in the PRANK
MSA, the corresponding sequences are spread out in a wide range of columns. These
examples show that the inferred evolutionary scenarios can be completely different
depending on the alignment methods used to analyze sequences.
6. Using simulated sequences for testing MSA methods
In this section we will discuss the use of simulation data in the comparison of alignment
methods. The advantage of using simulated sequences is the availability of the 'true'
alignment. In the simulation example discussed in this section, we simulate two sets of eight
lipocalin sequences described in Section 3. The lipocalin protein family has a common
structural feature, a single eight-stranded antiparallel beta-barrel. They also share three
conserved motifs. We will use the simulation program indel-Seq-Gen version 2.1 (iSGv2.1;
Strope et al., 2009) to simulate this lipocalin family proteins. iSGv2.1 is included in the
SuiteMSA package and the simulation can be done using its graphical user interface.


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Fig. 11. Comparison of gp120 alignment regions generated by MAFFT and PRANK. For both
panels A and B, the MAFFT MSA is used as the reference (top) and the PRANK MSA
(bottom) is compared against. The blue selection bar for the MAFFT MSA shows the
alignment area selected, and the green range bar for the PRANK MSA shows the column
range where corresponding amino acids are found in the MSA. The corresponding amino
acids in the two MSAs are shown with red color ('red' indicates that the alignment of these
characters is inconsistent between the MSAs). See the main text for the description on the
sequences marked with light blue and light green boxes.


231
6.1 Setting up the iSGv2.1 simulation
iSGv2.1 simulation requires a guide tree and a root sequence or MSA. By providing a root
MSA, instead of generating a random root sequence, the site-specific amino acid (or
nucleotide) frequency distribution derived from each MSA column can be used to generate a
simulation root sequence (for details, refer to iSG user manual). For the root MSA, we used
the 8-protein alignment of the lipocalin family we described in Section 3. The evolutionary
parameters chosen to simulate the lipocalin protein family are listed below. We performed
two simulations: the second more divergent than the first. For any parameters not
mentioned, default values were used. Three input files are prepared: a guide tree file, a
lineage file, and a root MSA file. For details on preparing the guide tree, the three motifs
used, and how to set up the length-limitation template, refer to Strope et al. (2009) as well as
Anderson et al. (2011). All input files used for this simulation are available from:
http://bioinfolab.unl.edu/~canderson/SuiteMSA/supplement.html.
i. Basic parameters
- Guide tree file: lipo8_3.tre (provides the guide tree and option parameters listed
below)
- Substitution model: PAM
ii. Advanced parameters
- Lineage file: lipo8_3.spec (provides the motif and template information)
- Branch scale: 0.5 (first simulation), 2.0 (second simulation)
- Random number seed: 6262
iii. Guide tree options (information included in the guide tree file)
- Use root msa file: lipo8_3template.root_in
- Maximum indel length: 10
- Insertion probability = deletion probability = 0.02 (first simulation), 0.025 (second
simulation)
- Indel length distribution = deletion length distribution: file name = inDL (provides
indel length distribution)
After running each simulation, we obtained a set of eight simulated sequences, the true
alignment of the eight sequences, and a record of all insertion and deletion events. As
shown in Fig. 12, the 'true' alignments from both simulations (the first more conserved and
the second more diverged) maintained the three conserved lipocalin motifs (M1, M2, and
M3) specified in the simulations. As expected, the second MSA derived from the simulated
sequences with a higher rate of substitutions (longer branch lengths) and a higher rate of
indel probability is about 100 aa longer (Fig. 12B, 303 aa) than the first MSA (Fig. 12A, 215
aa). We used these 'true' alignments as the references for the next analysis.
6.2 Comparison of MSA reconstruction using simulated sequences
We used four MSA methods (MAFFT, MUSCLE, Probalign, and PRANK) to align both
sets of the eight simulated sequences. For PRANK, the simulation guide trees (with
branch lengths scaled for the 'more conserved' and 'more divergent' simulations) were
used as the input phylogenies. In Fig. 13, the Pixel Plot is used to compare the
reconstructed MSAs against the reference MSAs (the true alignments obtained from the
two simulations). Tables 4 and 5 summarize the alignment statistics for the two sets of
simulated data.


232

Fig. 12. 'True' alignments of the two sets of simulated lipocalin protein sequences (A: more
conserved and B: more divergent simulations). Both alignments clearly show that the three
motifs (M1, M2, and M3) are conserved among these two sets of simulated protein
sequences.


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Fig. 13. Comparison of simulated lipocalin protein MSAs. The Pixel Plot is used to compare
four reconstructed MSAs with the reference alignments (A: more conserved and B: more
divergent simulations). The 'true' alignments obtained from the simulations are used as the
reference alignment (MSA 1). M1 (red box), M2 (blue box), and M3 (green box) show the
location of three conserved regions. The regions highlighted in magenta show an example of
inconsistent alignments found in reconstructed alignments relative to the true reference
alignments. MSA methods used are MAFFT (MSA 2), MUSCLE (MSA 3), Probalign (MSA 4),
and PRANK (MSA 5).
Fig. 13A shows that all four methods produced highly consistent MSAs for the sequences
obtained from the more conserved simulation. While two of the three conserved motifs were
identified correctly in all MSAs, in the region of the first motif (M1), all reconstructed MSAs
contained gaps. Consistently very high SPS' (0.91~0.93, Table 4) indicate that all methods
performed very well. The proportion of gaps is also consistent between all reconstructed
MSAs and the reference (~10%, Table 4).


234
Method SPS
CS with
no-gap
CS with
gaps
%
consistency
MSA
length
(aa)
%
conserved
% gaps
% no-gap
columns
Reference - - - - 215 7.4 10.9 74.9
MAFFT 0.933 0.850 0.782 72.77 213 7.0 10.0 75.1
MUSCLE 0.921 0.817 0.751 70.28 212 7.1 9.6 77.4
Probalign 0.912 0.787 0.704 63.76 218 7.8 12.1 73.9
PRANK 0.932 0.826 0.776 71.5 215 7.5 10.5 75.2
Table 4. Alignment statistics for the simulated 'more conserved' lipocalin family MSAs. SPS,
CS, and % consistency are obtained against the reference alignment. The highest value in
each comparison is shown in boldface.

Method SPS
CS with
no-gap
CS with
gaps
%
consistency
MSA
length
(aa)
%
conserved
% gaps
% no-gap
columns
Reference - - - - 303 2.00 39.70 39.60
MAFFT 0.533 0.481 0.263 20.64 252 2.40 27.50 42.90
MUSCLE 0.532 0.421 0.314 28.11 219 2.70 16.60 63.00
Probalign 0.585 0.465 0.321 23.56 258 2.30 29.20 49.20
PRANK 0.504 0.395 0.264 24.88 213 2.80 14.30 60.60
Table 5. Alignment statistics for the simulated 'more divergent' lipocalin family MSAs. SPS,
CS, and % consistency are obtained against the reference alignment. The highest value in
each comparison is shown in boldface.
In Fig. 14, we compared PRANK and MAFFT alignments more in detail using the MSA
Comparator. This is the same region highlighted in magenta color in Fig. 13A. The
alignment columns that are fully consistent between the reference and PRANK (Fig. 14A)
or MAFFT (Fig. 14B) are shown with blue color. Black characters, on the other hand,
indicate inconsistently aligned columns. For example, the characters contained in the red
square in the reference MSA are aligned exactly the same in the PRANK MSA. However,
as shown in Fig. 14B for the MAFFT MSA, the gap in column 35 (in the reference) is filled
with the characters shifted from the left. The Pixel Plot in Fig. 13 shows that the same
shifting and filling of the gap happened in all but the PRANK MSA. This demonstrates
the "gap magnet" phenomenon described in the previous section. Using the simulated
data, we know the origins of gaps. In Fig. 14, '-' in yellow cells are derived from deletion
events. Characters in green cells, on the other hand, are derived by insertion events.
Therefore, stacking up the 'QVD' sequences and avoiding inserting gaps as done by
MAFFT (Fig. 14B) is evolutionary incorrect. With commonly used affine-gap penalty
systems, opening new gaps is highly penalized as opposed to extending an existing gap.
This is reinforced with progressive alignment methods. This situation is clearly illustrated
in the example shown in Fig. 14B. Using its "phylogeny-aware" gap handling, PRANK
was able to correctly align these gaps.


235

Fig. 14. Comparisons of PRANK (A) and MAFFT (B) alignments against the reference
alignment (the simulated 'true' alignment). The region is taken from the area highlighted in
magenta in Fig. 13A. The MSA Comparator is used to show the actual insertion (marked
with green) and deletion (marked with yellow) events in the reference alignment. These
events are traced during the iSGv2.1 simulation.
When the divergence level was much higher, as shown in Fig. 13B, all methods could still
identify all of the three conserved motif sites. However, all MSAs were highly inconsistent
within the unconstrained areas. SPS' are significantly lower (0.50-0.59, Table 5). For this
dataset, PRANK produced the most inconsistent MSA, which was expected as PRANK is
recommended for aligning closely related sequences. It should also be noted that there is
little agreement among the alignments. This indicates that regardless of the statistics used,
no one method can be concluded as ideal. Using multiple methods is recommended so that
a selection of alignment hypotheses can be used to generate a more robust hypothesis.
7. Aligning ribosomal DNA sequences
We have so far concentrated our discussion on protein sequence alignments. In order to
obtain the full picture of alignment issues, in this section, we will examine the alignment of
ribosomal DNA (rDNA) sequences.
7.1 Small-subunit ribosomal DNA sequences and secondary structure
The ribosomal RNA genes contain large stretches of highly conserved sites (stem or knot
binding sites) interspersed with regions of varying sites (loop regions). These two types of
regions within the gene have different information content due to strong selective
constraints on the secondary structures and function within the stem and knot areas versus
very weak constraints on the loop area. Fig. 15 shows a predicted secondary structure of the
small-subunit ribosomal RNA (or 18S rRNA) from a parasitic protozoa Toxoplasma gondii, a
member of the family Sarcocystidae (Phylum Apicomplexa; Class Conoidasida; Subclass
Coccidia).
Figs. 16 and 17 show part of the 18S rDNA MSA of 60 Coccidia species (D. A. Morrison
personal communication; Morrison, 2009a). As shown in Fig. 16, stem regions are highly
conserved. This alignment illustrates the high level of conservation found in approximately
45% of the 18S rDNA alignment. On the other hand, large loop regions as the one shown in
Fig. 15 have much lower functional constraints. As shown in Fig. 17, sequences of such
regions are highly variable and alignment reconstruction of such regions often requires


236
laborious manual adjustment, iteratively incorporating information from the predicted
rRNA secondary structures (Morrison, 2009a).

Fig. 15. Predicted secondary structure of the Toxoplasma gondii 18S rRNA. The secondary
structure was obtained from Comparative RNA Web Site
(http://www.rna.ccbb.utexas.edu/; Cannone et al., 2002). The callouts 'S' and 'V' show the
'stem' and the large 'loop' regions, respectively. Their sequence-structure alignments are
shown at the bottom (the orange arrows pointing to the beginning and ending of the
regions).


237

Fig. 16. Alignment of a highly conserved stem region of 18S rDNA from 60 Coccidia species.
Using the MSA Viewer, the rRNA secondary structure information from T. gondii is
displayed below the alignment. This alignment corresponds to the region in the callout 'S'
shown in Fig. 15. The sites that are considered to be ambiguously aligned for this family are
indicated by a red 'A' in the structural representation. These positions do not appear in the
T. gondii structure. The alignment was provided by D. A. Morrison.
7.2 Comparison of 18S rDNA MSA reconstruction
We generated the alignments of full 18S rDNA sequences using four MSA methods. Using
the above-mentioned alignment provided by D. A. Morrison as the reference, we compared
the performance of the MSA methods. The alignment statistics are summarized in Table 6.

Method SPS
CS with
no gap
CS with
gaps
%
consistency
MSA
length
(nuc)
%
conserved
% gaps
% no-gap
columns
Reference - - - - 2095 50.60 15.80 62.40
Probalign 0.953 0.919 0.863 65.96 2389 44.30 26.10 55.60
MAFFT 0.950 0.898 0.844 73.08 2088 50.10 15.50 64.10
MUSCLE 0.950 0.917 0.867 73.77 2116 50.20 16.60 63.00
ClustalW2 0.948 0.946 0.855 75.23 2055 51.40 14.10 62.50
Table 6. Alignment statistics for the 18S rDNA MSAs. SPS, CS, and % consistency are
obtained against the reference alignment. The highest value in each comparison is shown in
boldface.


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Fig. 17. Alignment of a highly variable loop region of 18S rDNA from 60 Coccidia species.
One of the secondary structures used to refine the alignment was from T. gondii. This
structure is displayed below the alignment. This alignment corresponds to the region in the
callout 'V' shown in Fig. 15. The alignment was provided by D. A. Morrison.
All four methods appear to have produced alignments highly consistent with the reference.
This must be owing to highly conserved stem or functional regions that cover almost 50% of
the sequence regions. Such consistency is reflected by the high CS values particularly when
gapped columns are excluded (CS with no gap) and also the small differences in CS values
among MSAs. ClustalW2 has the highest un-gapped CS, indicating that ClustalW2 has the
highest number of columns that match the reference alignment, and likewise, the highest %
consistency. While the ClustalW2 MSA is the shortest (2,055 nucleotides), the longest and
most 'gappy' MSA was obtained by Probalign. Similar trends are found in the other
examples described in this chapter. Note, however, that the Probalign MSA had the highest
SPS (0.953) and second highest 'CS with gaps' (0.863; MUSCLE had a slightly better score,
0.867).
Now let us visually examine these alignments. Keep in mind that the sequences are highly
conserved, and that phylogenetic information will be derived mainly from the regions that are
sufficiently variable. In Fig. 18, the Pixel Plot was used to compare the four reconstructed
MSAs against the reference MSA. The selected area of the reference MSA under the blue
selection bar includes the subsequence shown in the callout 'V' of Fig. 15 (Fig. 16 also shows
the same area of the reference MSA). The magenta-colored pixels show the distribution of
characters included in this selected area. In the reference MSA, the magenta-colored area has
relatively small amount of gaps, providing the largest aligned overlap, putatively the most
phylogenetically informative region, within this large loop region. However, in the alternative
MSAs (MSAs 2-5), magenta-colored corresponding characters are spread over much wider
regions (green bars show the ranges covered by corresponding characters). Note that each
MSA method found a few conserved subsequences (matched columns) within this region.


239
However, each method also introduced a large number of gaps, affecting the consistency in the
alignments of the surrounding areas immediately before and after the selected region. In spite
of the high SPS observed with these MSAs and the degree of conservation within the
alignments, there is little consensus among the MSAs of this phylogentically critical area.
Through visual comparisons among alternative MSAs it becomes possible to recognize that
very different hypotheses could emerge depending on the MSA chosen. Such significant
differences among the MSAs are, and should be, alarming to researchers, since such
inconsistency in MSAs could affect phylogenetic hypotheses.

Fig. 18. Comparison of 18S rDNA MSAs. Pixel Plot is used to compare four reconstructed
MSAs with the reference. The alignment provided by D. A. Morrison was used as the reference
and compared with MSAs generated by Probalign, MAFFT, MUSCLE, and ClustalW2.


240
8. Conclusion
Advancements in the field of bioinformatics and molecular evolution have resulted in many
different methods for reconstructing MSAs. While each MSA method has a different objective
function and different heuristics to maximize the objective function for building the alignment,
if they were in fact meant to reconstruct alignments that reflect the evolutionary history of
sequences, we would expect some level of consensus between them. Such is not the case in
reality. We used five types of alignment problems in this chapter. Using seven different MSA
methods, we discussed the similarity and difference among MSAs built by these methods. We
have shown that assessment of MSAs can be performed using a combination of descriptive
statistics both for individual alignments and the comparison of two alternate alignments. We
have also shown that using visual tools provided by SuiteMSA, we can examine MSAs based
on the alignment of structural features such as secondary structure and transmembrane
predictions. We further demonstrated how the sequence simulator included in SuiteMSA can
be used to produce benchmark alignments.
We should keep in mind that alignments reconstructed by any MSA methods are only
hypotheses on the evolutionary relation of the sequences. Furthermore, while these alignments
can be assessed as consistent (or not) with the accepted model for the given sequences (the
reference alignment), this reference is itself a hypothesis unless generated by a simulation
program and may not be 'correct'. It is important for the researcher to understand the
underlying assumptions of the alignment methods as well as the characteristics of the
biological sequences to be aligned and to assess the resulting alignments. User friendly
graphical tools such as SuiteMSA can assist in the critical assessment of MSAs prior to their
use in further studies.
9. Acknowledgements
We would like to thank Dr. David A. Morrison (Swedish University of Agricultural
Sciences) for providing us 18S rDNA alignments and discussion. This work has been
partially supported by NSF AToL grant 0732863 to ENM.
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12
Optimal Sequence Alignment
and Its Relationship with Phylogeny
Atoosa Ghahremani and Mahmood A. Mahdavi
Department of Chemical Engineering, Ferdowsi University of Mashhad,
Azadi Square, Pardis Campus, Mashhad,
Iran
1. Introduction
The main motivation for predicting functions of hundreds of thousands of genes and
proteins found across genomes and proteomes is variations within a family of related
nucleic acid or protein sequences that provide an unreliable source of information for
evolutionary biology. Protein molecules are more diverse in structure and function than any
other kind of molecule. Then if nucleic acid sequences undergo mutations, insertions,
crossing-over and some another changes, these variations have a direct effect on the coded
protein molecules (Fitch, 1970; Pearson et al., 1997). If a protein sequence is present in many
different organisms or be conserved along evolution, it is predicted that it might have a
similar function in all the organisms. Two molecules of related function usually have similar
sequences reciprocally two molecules of similar sequence usually have related functions
(Dardel, 2006). The objective of bioinformatics is to detect such similarities, using computer
methods to draw biological conclusions. Collecting available wealth of sequence
information, help to track ancient genes and back trough the tree of life then to discover new
organisms based on their sequences (Fitch, 1966). Searching diverse genes may show
different evolutionary histories that reflecting transfers of genetic material between species.
If we recognize the function and/or structure of a member of an evolutionary family then
we can predict the function of all the other members and even identify the important
functional groups. For this, we need to identify which proteins are belonging to the same
family and then distinguish proteins that are evolved from the same ancestor after a set of
accepted mutation events. Such proteins have amino acid sequences that are likely to be
more similar than expected for unrelated protein sequences. When two or more than two
sequences share a common evolutionary ancestor they called homologous (Fitch, 1970).
There is no homology degree, sequences are either homologues or not (Reeck et al., 1987;
Tautz, 1998). These types of proteins almost always share a significantly related tree-
dimentional structure. An example for very similar structures which is determined by x-ray
crystallography is RBP and -lactoglobulin (Fig. 1). Once the homology between some
related sequences is inferred, identity and similarity are the quantities for describing the
relatedness of sequences. In one type of homology, two sequences may be homologous but
without sharing statistically significant identity. In general, three dimensional structures
differ much more slowly than amino acid identity between two proteins (Chothia & Lesk,


244
1986). There are two types of homology, orthology and paralogy. Orthologs are homologous
sequences that are in different species but arose from a common ancestral gene during
speciation event. It has been predicted that orthologous sequences have similar biological
functions (In Fig. 2, human and rat RBPs both transport vitamin A in serum). Paralogs are
homologous sequences evolved from gene duplication mechanism. An example for
paralogous sequences is human RBP plasma to the other carrier protein human
apolipoprotein D (Fig. 3). It is predicted that paralogous sequences have distinct functions
but their functions are related together (Pevsner, 2003a; Mount, 2001a).
Homology inference heavily relies on alignment of primary structure of proteins and DNA
sequences. This is a procedure for identifying the matching residues within the sequences
sharing the same functional and/or structural role in the different members of the family
(Xu & Miranker, 2003). After performing alignment and evaluating alignment scores, the
most closely related sequence pairs become apparent and may be placed in the outer
branches of an evolutionary tree. With continuing alignment procedure for different
sequences of particular gene, a predicted pattern of evolution for that particular gene is
generated and a tree has been found for inferring the changes that have taken place in the
tree branches. Therefore, the first step for making a phylogenetic tree is a sequence
alignment (Feng, 1985). An indication for each pair of sequences is the sequence similarity
score. A tree is derived based on the best accounts for the numbers of changes (distances)
between the sequences of these scores.

Fig. 1. Tree-dimentional structure of two lipocalins: bovine RBP (left side), bovin -
lactoglobuline (right side). These two proteins are homologous (evolve from a common
ancestor), and they share very similar tree-dimensional structure consisting of a binding
pocket for a ligand and eight antiparallel beta sheets.

Optimal Sequence Alignment and Its Relationship with Phylogeny

245

Fig. 2. Orthologous RBPs. In this tree, sequences that are more closely related to each other
are grouped closer.

Fig. 3. Paralogous of human lipocalin proteins. Each of them is a member of protein family.


246
2. Alignment approaches
Sequence alignment is a way for comparing two (pair-wise alignment) or more than two
(multiple alignment) sequences. This procedure looks for a series of particular residues or
patterns that are in the same order. It is useful for discovering functional, structural, and
evolutionary information in biological sequences (Wen et al., 2005; Berezin et al., 2003;
Smoot, 2003). After sequence analysis if very much alike or similar sequences are found,
they will probably have the same or similar biochemical functions and tree-dimensional
structures (for protein sequences). If two sequences from different organisms are similar,
there may have evolved from a common ancestor and the sequences are then defined to be
homologous (Doolittle, 1981; Fitch & Smith, 1983; Feng & Doolittle, 1985). There are two
approaches for sequence alignment: multiple sequence alignment and pair-wise sequence
alignment.
2.1 Multiple sequence alignment
Multiple sequence alignment is a widely used method for comparing subsequences or entire
length of more than two sequences and discovering the relations of their host organisms
(Fig. 4). If two sequences are very close in terms of evolution, most of their residues remain
unchanged and it will be rather difficult to detect important residues. On the other hand, if
two sequences are evolutionarily distant, a reliable alignment of their sequences will be
much more difficult to obtain. With aligning highest number of sequences of homologous
proteins the aforementioned problem will be solved. Performing alignment the highly
conserved residues that define structural and functional domains in protein families will be
identified. New members of these families with the same domains can be found by
searching sequence databases. A multiple sequence alignment implies a pair-wise alignment
for each pair of sequences. The score of the multiple sequence alignment is the sum of scores
of all implied pair-wise alignments. Multiple sequence alignment often tells us more than
pair-wise alignment because it is more informative about evolutionary conservation (Edgar
& Sjolander, 2004). The most common algorithm for multiple sequence alignment is BLAST.
This algorithm has some programs like CLUSTALW for performing alignment and
CLUSTALX for preparing graphical representation of the alignment (Larkin et al., 2007)
2.2 Pair-wise sequence alignment
In pair-wise alignment, two sequences are placed directly next to each other in two rows.
For aligning protein sequences, the single-letter amino acid code is used. Identical or similar
residues are placed in the same columns and non-identical residues can be placed either in
the same column as a mismatch or opposite to a gap in the other sequences. The gaps are
introduced to the sequences for shifting the residues (without disturbing its order) and
obtaining the most possible matched residues, also for generating sequences with the same
lengths. Some similar not identical residues are identified by pair-wise sequence alignment.
Similar pairs of residues are related to each other because they share similar biochemical
properties and are related functionally and structurally. When two similar residues are
aligned, it is a representation of a conservative substitution that occurred during evolution.
Amino acids with similar properties are comprised acidic amino acids like "D, E", basic
amino acids like "K, R, H", hydroxylated amino acids "S, T", and hydrophobic amino acids
"W, F, Y, L, I, V, M, A" (Pevsner, 2003a).


247

Fig. 4. Multiple sequence alignment of the portion of the glyseraldehyde 3-phosphate
dehydrogenase (GAPDH) protein from six organisms.
For homology inference, after aligning two sequences some quantities must be calculated
including percent identity and percent similarity. The percent similarity or positive of two
protein sequences is the sum of both identical and similar matches divided by length of
alignment and characterized with mark (:) in the alignment. The percent identity is
concluded from the number of identical residues divided by the length of alignment and is
shown with (|) mark in the alignment (Fig. 5). Since the similarity measure is calculated
based upon a variety of definitions for identifying the degree of related residues, then it is
more useful to consider the degree of identity shared by two protein sequences. In aligning
sequences with different lengths, there must be no column with merely gap characters. In an
optimal alignment, mismatched residues and gaps are placed in positions where bring as
many as possible identical and similar residues.
2.2.1 Gaps and gap penalties
For obtaining the best possible alignment, introducing gaps in alignment and gap penalties
for calculating alignment score is necessary. The addition of gaps in an alignment may be
biologically relevant because the gaps reflect evolutionary changes that have occurred. They
also allow full alignment of two proteins. The gaps represent two of tree types of common
mutations occurred during evolution and caused divergence of the sequences of the two
proteins. Insertions and deletions occur when residues are added or removed during
evolution relative to the ancestor protein sequence and cause entering null characters or
gaps to one of the sequences while aligning. There are two types of gap penalties: gap
opening penalty for any gap (g) and gap extension penalty for each element in the gap (r)
(Resee, 2002; Edgar, 2009). Thus, the total gap score w
x
can be calculated.

x
w g rx = + (1)


248
where, x is the length of the gap. There are several forms of gap penalty, including: 1-
constant penalty, the simplest form where each gap is given a constant penalty independent
of the length of the gap, 2-proportional penalty where the penalty is proportional to the
length of the gap. With this form, longer gaps are given higher penalties than shorter ones,
3-affine gap penalty that is the most complex form of gap penalty (Fig. 6). It has both
constant and proportional contributions. The motivation for using affine gap penalty is that
opening a gap should be strongly penalized, but once a gap is opened it should cost less to
extend it. If the used gap penalty is too high relative to the range of scores in the substitution
matrix, gap will never appear in the alignment, but conversely if the gap penalty is too low
compared to the matrix scores, gaps will appear everywhere in the alignment in order to
align as much same residues as possible.

Fig. 5. Pair-wise alignment of human RBP and -lactoglobulin. The alignment is global (the
entire lengths of each protein is aligned) and there are many positions of identity between
two sequences (shown with |). Dots are different. (1) The pair dots indicating different
amounts of similarity (like R and K that share similar biochemical properties). (2) Single dots
also indicate similarity, but less than paired dots. (3, 4) Dots in the place of alphabetic
characters along the sequences show internal and external gaps. (5) A dot indicated above
the sequences entered for marking every 10 residues.


249

Fig. 6. A typical illustration of calculating gap affine penalty.
2.3 Alignment algorithms
For short and very closely related sequences, finding the best alignment is easy. However, in
cases where sequences are long and not closely related finding the best alignment is rather
difficult. If gaps are introduced in the alignment to account for deletions or insertions in the
two sequences, the number of possible alignments increases exponentially. In these cases,
computational methods are required. The known computational methods for this task are
called dynamic programming algorithms. Such algorithms take two input sequences and
produce the best alignment between them as output (Sankoff, 1972).
In general, there are two approaches for aligning sequences, global alignment and local
alignment. In global alignment, the entire length of the sequence is subject to alignment.
Sequences that are quite similar and their lengths are approximately the same are suitable
for global alignment. In local alignment, the subsequences with the highest number of
identical or similar residues are aligned and generate an alignment that is terminated at the
ends of the regions with strong similarity. This type of alignment is a suitable way for
aligning sequences that are similar along some regions of their length but dissimilar in
others, sequences with different length, and those sequences share conserved regions. In
sequence similarity analysis two dynamic programming algorithms are commonly used, the
Needleman-Wunsch algorithm and the Smith-Waterman algorithm. These algorithms are
closely related, but the main difference is that the Needleman-Wunsch algorithm finds
global similarity between sequences while the Smith-Waterman algorithm finds local
similarity. The Smith-Waterman algorithm is the most used, because in reality biological
sequences are not often similar over their entire lengths, but are similar only in particular
regions (Pearson, 1992; Smith & Waterman, 1981a; Smith et al., 1981b).
2.3.1 Global sequence alignment
Needleman-Wunsch algorithm is one of the first and most important algorithms for aligning
two protein sequences based upon dynamic programming. The importance of this algorithm
is from the point that it produces an optimal alignment of protein or DNA sequences even
with entering the gaps. Generating global sequence alignment using this algorithm
undergoes tree steps: 1-setting up identity matrix, 2-scoring the matrix, and 3-identifying the
optimal alignment. In the first step, the two sequences are placed in a two-dimensional


250
matrix (Fig. 7). The first sequence of length "m" is arranged horizontally along x axis so that
each amino acid residue correspond to a column. The second sequence of length "n" is listed
vertically along the y axis so that each amino acid residue corresponds to a row. For
generating an amino acid identity matrix, simply each cell takes a value of +1 if the
corresponding residues in row and column are identical and zero otherwise. Thus, for two
identical sequences, in this matrix the +1 value would describe a diagonal line from top left
to bottom right.
In the second step, a scoring matrix is generated. The assignment of scores starts from the
bottom right of the matrix, corresponding to the carboxy termini of the proteins, and
proceeds to the top. For moving through the matrix, to define a path corresponding to the
sequence alignment, there are several rules. Briefly, for setting up the scoring matrix in the
second step, at position i and j, take the value of the cell plus the maximum score obtained
from any of the following three values:
1. The score diagonally down (at position i+1, j+1), without including any gaps.
2. The highest score may find in position i+1, j+2 to the end of row j. Finding the highest
score in this position cause to the addition of a gap in the column. The number of gap
can be greater than 1.
3. The highest score may find in position i+2, j+1 to the end of column of i. This finding
corresponds to the addition of a gap in the row.
The third step is identifying the optimal alignment, i.e. the path through the matrix that
maximizes the score. Thus, a path through as many positions of identity as possible while
introducing as few gaps as possible must be found exploiting a trace-back strategy. We
begin at the upper left of the matrix (amino termini of the proteins) with the highest value
(in Fig. 7 this value is "+8" corresponding to an alignment of residues A to A). Then we find
the path down and to the right with the highest numbers along the diagonal. Going off the
diagonal implies automatically the insertion of a gap in one of the sequences and entering
some penalty. There may be more than one optimal alignment where all of them have an
equally high score (Fig. 7). In such cases that uses unitary scoring scheme, multiple optimal
alignment is obtained, but the introduction of a sophisticated scoring matrix like series of
BLOSUM and PAM, it is unlikely to find multiple optimal alignments. For evaluating the
obtained global alignment, the percent identity and similarity shared by two proteins, the
length of the alignment, and the number of gaps which is introduced to the alignment is
calculated (Needleman & Wunsch, 1970).
2.3.2 Local sequence alignment
Local alignment, a modified dynamic programming algorithm, seeks the highest scoring
local match between two sequences. This algorithm proposed by smith and waterman (1981)
is a very strong method for finding the high scoring subsets of two protein or DNA
sequences. It is very useful in a variety of applications such as database searching. In
general, this algorithm generates a matrix by two protein sequences and then finds the
optimal path along a diagonal like global algorithm, but the alignment does not necessarily
extend to the ends of the two sequences and for starting the alignment from some internal
position, there is no penalty.
The Smith-Waterman algorithm constructs a matrix with an extra row along the top and an
extra column on the left side. Thus, for two sequences of lengths "m" and "n", the matrix
dimension is m+1 by n+1. The score of each cell is selected as the maximum score in the


251

Fig. 7. Global pair-wise alignment of two amino acid sequences using a dynamic
programming algorithm. Generating the scoring matrix and using the trace-back procedure
for obtaining the optimal alignment path is shown and ultimately the alignment of the two
equally optimal path are shown in section d (the upper path) and e (the lower path).
preceding diagonal or the score obtained from the introduction of a gap, but the score
cannot be negative. In this algorithm if a negative value is generated in each cell, a zero is
inserted in the cell, instead (Fig. 8). The score of each cell like i, j or H(i, j) is given as the
maximum of four possible values:
1. The score which is located at position i-1, j-1 (the score diagonally up to the left). This
score is added to the new score in position s(i, j) which consists of either a match (1) or a
mismatch (-0.3).
2. s (i, j-1), located at one cell to the left minus a gap penalty.
3. s (i-1,j), immediately above the new cell, minus a gap penalty.
4. zero. Assures that there is no negative value in the matrix.
For two sequences, a=a
1
a
2
a
n
, and b= b
1
b
2
b
m
, where H
i,j
= H (a
1
a
2
a
i
, b
1
b
2
b
j
),
then:

, 1, , ,
max{ 1 ( , ), max( ), max( ), 0}
i j i j i x j x i j y y
H H s a b H w H w

= + (2)
0 0
0
x y
H H = = for 0 x n s s and 0 y m s s
1 i n s s and 1 j m s s


252

1
1
3
x
w
x
= +
and
1
1
3
y
w
y
= +
(3)
In equation (2), H
i,j
is the score at position i in sequence a and position j in sequence b, s(a
i
,
b
j
) is the score for aligning the characters at positions i and j. In equation (3), w
x
is the
penalty for a gap of length x in sequence a, and w
y
is the penalty for a gap of length y in
sequence b.
The maximal alignment can begin and end everywhere in the matrix so that the linear order
of the two amino acid sequences cannot be violated. The trace-back procedure finds the
highest value in the matrix and begins the alignment from the position of the highest
number. It proceeds diagonally up to the left until a cell is reached with a value of zero. The
zero value defines the start of the alignment, and is not necessarily at the extreme top left of
the matrix (Smith & Waterman, 1981).

Fig. 8. A typical example for pair-wise local sequence alignment using smith-waterman
algorithm.
3. Rapid and heuristic versions of smith-waterman: FASTA and BLAST
Theoretically sequence alignment techniques are based upon two different backgrounds
(Pearson, 1996, 1988): Dot matrix analysis ( Gibbs & McIntyre, 1970) and the dynamic
programming analysis such as Needleman-Wunsch and Smith-Waterman. The dot matrix
analysis is used when the sequences are known to be very much alike and this similarity is
clearly observed by displaying any possible alignments as diagonals on the matrix. This
analysis reveals readily any insertions, deletions, direct and inverted repeats that are found
with difficulty by the other methods. However, major limitation of this analysis is that most
of these programs do not show an actual alignment. For comparing sequences based on this
analysis, one sequence (A) is listed across the top of a page and the other sequence (B) is
listed down the left side. Starting with the first character in sequence B and then move
across the page to the end of the first row and placing a dot in any column where the
character in sequence A is the same. This continues until the page is filled with dots


253
representing all the possible matches of A characters with B characters. Any region of
similar residues is identified by a string of dots located on the diagonal. Other dots, located
on the positions everywhere other than diagonal represent random matches that are
probably not related to any significant alignment.
There are tree types of variations for analysis of two protein sequences by the dot matrix
method. First, one can use chemical similarity of the amino acid R group or some other
features for detecting similarity score. Second, one can apply the specific scoring matrices
such as PAM and BLOSUM. These matrices provide scores for matches that have occurred
based on aligning the protein families (these matrices will be described in section 4) (States
& Boguski, 1991). Finally, it can be analyzed by producing several different matrices, each of
them with a different scoring system and with average of different scores. This method is
suitable for more distantly related proteins.
Although the alignment algorithms based on dynamic programming analysis such as Smith
and Waterman guaranteed to find the optimal alignment(s) between two sequences, it is
relatively slow. For pairwise alignment, the speed is not a problem but when it is used for
database searching, that is, comparing one sequence as a query to an entire database, the
speed of the algorithm becomes an important factor. In most algorithms there is a parameter
called N that refers to the number of data items need to be processed. The required time for
the algorithm to perform a task is greatly affected by this parameter. If the running time is
proportional to N, then doubling N doubles the running time. For both algorithms based on
dynamic programming, Needleman-Wunsch and Smith-Waterman, the memory space and
the time required for aligning two sequences is proportional to the product of the length of
two queries, mn, and for the search of a database of size N, that is, mnN. The modified
algorithm of Smith-Waterman was developed to provide rapid alternative algorithms such
as FASTA (Pearson and Lipman, 1988) and BLAST (Basic Local Alignment Search Tool)
(Altschul et al., 1990). Both of these algorithms require less time to perform an alignment.
These algorithms are heuristic and since they restrict the search by scanning a database for
likely matches before performing the actual alignment they require less time, but it is not
guaranteed to find optimal alignments.
3.1 FASTA heuristic algorithm
This algorithm, divides the query sequence as well as the considered database into
subsequences with arbitrary lengths (for protein sequences two or three amino acid
length), so called words. Then, the positions of the words in the query sequence and
database sequences are calculated. The ktup value or the length of the words is a value
which determines how many consecutive identities are required for a match to be
declared. The lesser the ktup value, the more sensitive the alignment. Often, ktup = 2 is
taken for proteins, and ktup=6 for nucleotides. The same word can appear more than once
in the sequence without affecting the algorithm (Pearson, 2000). After dividing sequences
according to ktup value to consecutive subsequences, the relative position of each word in
the two sequences is calculated by subtracting the position of the word in the query from
each of the database sequences. Those words that have the same offset, they can be part of
the same alignment without insertions or deletions. Therefore, by constructing a look-up
table, all dense regions of identities between two sequences are identified. Next, the score
of each aligned regions is calculated using PAM250 matrix selecting the 10 highest scoring
regions for each database sequence. The sum of the scores of the 10 regions is called the
best initial regions (init1) and used to rank the matches for further analysis. The longer


254
regions of identity are generated by joining initial regions (initn) with scores greater than
a certain threshold. The initn score is the sum of the scores of these aligned regions after
subtracting a penalty accounting for the gaps. In later versions of FASTA, an optimization
step is added. When the initn score reaches to a certain threshold value, the score of the
region is recalculated for producing an OPT score by performing a full local alignment of
the region using Smith-Waterman dynamic programming algorithm. This optimization
increases the sensitivity but decreases the selectivity of the search (pearson, 1990,
1991,1998; Tramontano, 2006; Mahdavi, 2010). These scores (initn and OPT) are the basis
to rank database matches.
3.2 BLAST heuristic algorithm
The BLAST algorithm was established as a new tool to perform a sequence similarity search
based on an algorithm that is faster than FASTA, but is as sensitive as FASTA. The BLAST
web server (http://www.ncbi.nlm.nih.gov) is the most widely used for sequence database
searches and is backed up by a powerful computer system. The original version of the
BLAST looks for contiguous similarity regions between the query and database sequences
(without using gaps). The speed of the algorithm like FASTA increases by initially searching
common words or k-tuples in the query sequence and each database sequence. While
FASTA searches for all possible words of same length, BLAST searches the words that are
most significant. The word length for this algorithm is fixed at 3 for proteins and 11 for
nucleic acids. This length is the minimum length required to achieve a word score that is
high enough to be significant but not so long to miss short but significant patterns. There are
several steps involved for searching a protein sequence database for a query protein
sequence by BLAST algorithm (Altschul et al., 1990, 1994, 1997). In similarity searching by
BLAST program, three steps need to be taken. The program compiles a preliminarily list of
pair-wise alignment called word pairs. Then the algorithm scans a database for word
pairs that meet some threshold score T and extends the word pairs to find those sequences
that scores better than the cutoff score S. Scores are calculated from scoring matrices (such as
BLOSUM62) along with gap penalties.
In preprocessing stage, the query string is divided into words of length 3. The goal of the
preprocessing stage is to build a hash table, which is called query index. The keys of the
hash table are the 202020=8000 possible tree-letter words. The value associated with each
word is the position of that word in the list of all query words that gain a high score when
aligned against the key word. The threshold for high-score that is defined by default in
BLOSUM62 scoring matrix is 11. Threshold score or neighborhood word score threshold (T)
is selected for reducing the number of possible matches. For example, if a three-letter word
PQG occurs in the query sequence, the match score of this word to itself is calculated by the
log-odds BLOSUM62 matrix as P-P match, plus that for a Q-Q match, plus that for a G-G
match that equals to 7+5+6=18. Similarly, the PQG match to PEG scores 15, to PGR 14, to
PSG 13, and to PQA 12. For DNA words, the score for a match is +5 and for a mismatch is -4.
With selecting the threshold score, the list of possible matching words is shortened from
8000 (for w (word length) = 3) to the highest scoring words that satisfy the threshold score.
The preprocessing stage is repeated for each three- letter word in the query sequence. The
remaining high-scoring words that include possible matches to each three-letter position in
the query sequence are listed in a table called the query index in order to create an efficient
rapidly comparing search to the database sequences. In the second step, each database
sequence is scanned for identifying an exact match to one of the words listed in the query


255
index. If a match is found, this match is used to seed a possible ungapped alignment
between the query and database sequences. In the last step, an attempt is made for
extending an alignment from the matching words in each direction along the sequences. The
extending process is continued as long as the score is increased and is stopped once the
accumulated score did not increase and begun to fall a small amount below the best score
found for shorter extensions (Dawid, 2001; Pevsner, 2003b). In this condition, a longer
stretch of sequence (called the HSP or high-scoring segment pair) with a greater score than
the original word is found. In order to determining a suitable value for S, the range of scores
found by comparing random sequences is examined and significant values are selected. In
the later version of BLAST, called BLAST2 or gapped BLAST (Altschul et al., 1997; Brenner,
et al., 1998), a list of high-scoring matching words is made similar to the original method
with the exception that a lower value of T, the word cutoff score, is used. The lower cutoff
score produces longer word list and matches to lower scoring words in the database
sequences.
In order to remove the low-complexity regions that are not useful for producing meaningful
sequence alignments, the filtering programs is used. Filtering masks portions of the query
sequence that have commonly found stretch of amino acids or nucleotides with limited
information content. For protein sequence queries, the SEG program is used and for nucleic
acid sequences, the DUST program is employed. Using Filtering programs, low complexity
residues are replaced with a string of characters with the letter X (for protein sequences) or
N (for nucleic acid sequences). In general, filtering is useful to avoid receiving spurious
database matches, but in some cases authentic matches may be missed.
3.2.1 An example
Let the following query sequence:
C I N C I N N A T I (w=3, n=10, T=11, BLOSUM62 matrix)
where, the number of words with length 3 (w=3) is calculated as follows:
1 N n w = + (4)
Then, for the given query sequence, N=8. The three-letter words of the query sequences are:
C I N (1) I N C (2) N C I (3) C I N (4) I N N (5) N N A (6) N A T (7) ATI (8)
Using BLOSUM62 matrix, 54 words of 8000 key words in the hash table obtain score 11 or
greater when aligned with the C I N word which is located at positions 1 and 4.
CAN CCN CDN CEN CFN CGN CHN CIA CID CIE CIG CIH CIK CIM CIN CIP CIQ CIR
CIS CIT CIY CKN CLD CLE CLG CLH CLK CLN CLQ CLR CLS CLT CMD CMH CMN
CMS CNN CPN CQN CRN CSN CTN CVD CVE CVG CVH CVK CVN CVQ CVR CVS
CVT CWN CYN
Similarly, only three pairs obtain score 11 or greater when aligned with A T I at position 8.
Overall, preprocessing of the query sequence assigns 204 entries of the 8000 possible keys.
After preprocessing stage, the next step is scanning the target string (reference sequence)
successively for finding exact matches to one of the words in the query index. Suppose,
following sequence as a target string:
P R E C I N C T S


256
For this sequence N=7, then the three-letter words along their locations are:
PRE(1), REC(2), ECI(3), CIN(4), INC(5), NCT(6), CTS(7)
Looking up NCT at position 6 of the target string, the search generates hits (3,6) and (7,6).
This means that similar words to position 6 at the reference sequence are at positions 3 and 7
of the query sequence. After finding the location of the exact matches, each hit is extended to
the right and to the left to increase the alignment's score. The alignment is extended until the
overall alignment score maximizes. In this example, the corresponding alignment for the hit
at query position 3 and target position 6 is:
- - - c i N C I N n a t i
p r e c i N C T S - - - -
Hence, the final local alignment is:
C I N C I N
C I N C T S
The score of this local alignment is calculated as follows:
S
CC
+ S
II
+ S
NN
+ S
CC
+ S
IT
+ S
NS
= 9 + 4 + 6 + 9 + (-1) + 1 = 28
Another hit at query position 7 and target position 6 is:
c i n c i n N A T I
- p r e c i N C T s
The score of this alignment can no longer be increased by further extending it to either left
or right (Dwyer, 2003).
4. Representation of different substitution matrices
4.1 Amino acid substitution matrices
Amino acid arrangement of proteins and nucleic acids change due to mutations occur over
the course of evolution. Amino acids are substituted by other amino acids during mutation
and these substitutions cause variations in phenotype of the related species. There are some
regions in the sequence that undergo massive mutations and some other regions remain
conserved over a long period of time in evolution. The alignment outcome demonstrates
conserved regions in related protein sequences that represent functions of the proteins
(Campanella et al., 2003). Additionally, it shows some amino acid substitutions commonly
occur in related proteins from different species. Substituted amino acids are compatible with
protein structure and function and are chemically similar to amino acids which are changed.
Some substitutions are rare or least common and some of them are most common. Sequence
alignment is a useful tool for understanding the type of changes occurred in related protein
sequences. Based on the type of substitution different matrices were built such as PAM and
BLOSUM. Substitution matrices are used in sequence alignments while they are built out of
aligning carefully selected sequences. In the following the detail description of PAM and
BLOSUM substitution matrices is presented.


257
4.1.1 PAM (point accepted mutation) matrices
Margaret Dayhoff (1978) developed a method for determining the most likely amino acid
changes that occurred during evolution by assessing ancestral relationship among a group
of proteins (Kim & Kececioglu, 2008). The analysis was performed based on multiple
sequence alignment of 34 closely related protein superfamilies which were grouped in 71
phylogenetic trees (such as: cytochrome c, hemoglobin, myoglobin, virus coat proteins,
chymotrypsinogen, glyceraldehydes 3-phosphate dehydrogenase, clupeine, insulin and
ferredoxin). The studied groups of proteins ranged from very well conserved (like, histones
and glutamate dehydrogenase) to proteins with high rate of point mutations (like
immunoglobin chains and carrier proteins). In this model for creating the mutation data
matrix (MDM), the sequences of all of the nodal common ancestors in each tree were
generated by multiple sequence alignment of each protein family, then counting the most
frequent amino acids for inferring the common ancestor of each family from those most
frequent amino acids. The matrix of accepted point mutation was calculated for each protein
family separately from the constructed phylogenetic tree which was inferred for each
studied protein family. In this matrix it was assumed that the likelihood of amino acid X
replacing Y is the same as that of Y replacing X, and hence 1 was entered in cell YX as well
as in cell XY (Dayhoff, 1972). Dayhoff assumed that by considering this symmetry, the
frequency of occurrence of an amino acid in any large group of studied proteins appears to
have been relatively constant with time. The accumulated accepted point matrix for closely
related sequences was generated by summing the number of corresponding elements of
each separately accepted point matrix, which was computed for each protein family
sequences together. Next, the relative mutability of the 20 amino acids in sequences of each
studied protein family was calculated. Relative mutability was simply calculated as the
number of observed changes of an amino acid divided by its frequency of occurrence in the
aligned sequences. Mutability was normalized with respect to the basic unit of evolutionary
distance as being a single accepted point mutation in a sequence of length 100.
Consequently, the average relative mutability of an amino acid was therefore the total
number of changes observed for this amino acid in all the families of studied proteins,
divided by the total sum of all local frequencies of occurrence of the amino acid multiplied
by the numbers of mutations per 100 residues in each of the branches of all the family trees.
The mutation probability matrix was then constructed (Fig. 9). An element of this matrix,
H
]
, gives the probability that the amino acid in column j would be replaced by the amino
acid in row i after a given evolutionary interval. The values of the non diagonal elements of
this matrix were computed by following equation (Dayhoff, 1972):

j ij
ij
ij
i
m A
M
A
(5)
Where, A
]
is an element of the accepted point mutation matrix, z is proportionality
constant, and m
]
is the mutability of the j
th
amino acid. The values of diagonal elements are
calculated as follows:
1
ij j
M m = (6)
In mutation probability matrix, the ratio of the individual non-diagonal terms within each
column has the same ratio of the observed mutation in the mutation data matrix. The


258
proportionality constant z is the same for all columns of the matrix and is calculated by
following equation for 1PAM evolutionary interval in which 1% of amino acids have
changed (Higgs & Attwood, 2005):
0.01
tot
tot
N
A
= (7)
where, N
tot
is the total number of amino acids in the data set, and A
tot
is the total number of
elements in the A
]
matrix.
In mutation probability matrix, the diagonal elements are all slightly less than one, and off
diagonal elements are very small. The number of unchanged amino acids, when a 100-
residue protein sequence (of average composition) is exposed to the evolutionary changes, is
computed as follows:
100
i ii
i
f M
(8)

Fig. 9. Mutation probability matrix for the evolutionary distance of 2PAMs. Each element of
this matrix gives the probability that the amino acid in column j will be replaced by the
amino acid in row i after a given evolutionary interval, in this case 2 accepted point
mutations per 100 amino acids. Values are multiplied by 10000 for convenience.


259
Other PAM matrices are calculated from multiplying PAM1 matrix by itself with respect to
the characteristic number of the PAM matrix (e.g. PAM250 matrix is produced when the
PAM1 matrix is multiplied by itself 250 times). A scoring system has been developed for
converting the elements of a PAM mutation probability matrix into a scoring matrix or log-
odd matrix as follows (Pevsner, 2003a):

10
( , ) 10log
ab
b
M
S a b
P
| |
=
|
\ .
(9)
where, H
ub
is the probability that the aligned pair of amino acid residues a and b represent
an authentic alignment and
b
is the normalized frequency representing the probability that
the residue b was aligned by chance.
The PAM1 matrix is recalculated (Jones et al., 1992) using updated data, called PET91. This
dataset was generated from Release 15.0 of the SWISS-PROT protein sequence database
(Bairoch, 1996), containing 16941 sequences. Also, a mutation data matrix has been
calculated for transmembrane proteins (Jones et al., 1993). It was found that this new
mutation data matrix is very different from matrices calculated from general sequence sets
which are biased towards water-soluble globular proteins. The differences are discussed in
the context of specific structural requirements of membrane spanning segments. Calculating
the mutation data matrix for each protein family and hence creating specific PAM scoring
matrix for each protein family will help to improve the accuracy of the protein sequence
alignment results.
4.1.2 BLOSUM matrices (Blocks Amino Acid Substitution Matrices)
BLOSUM scoring matrices are improved alternatives to PAM. These series of scoring
matrices are widely used for scoring protein sequence alignments. The BLOSUM matrices
are derived from the database for storing the sequence alignments of the most conserved
regions of protein families, BLOCK database (S. Henikoff & J.G. Henikoff, 1996). This
database of blocks is consisted of over 500 groups of local multiple alignments of distantly
related proteins. The BLOSUM matrix values are obtained by the same method applied to
PAM matrices. The values are computed from the observed amino acid substitutions in a
large set of about 2000 conserved amino acid patterns. The constructed blocks from patterns
of amino acids in each protein family, derived ungapped multiple alignments. For deriving
BLOSUM matrices from blocks, not all the sequences are used but a percentage of identity
higher than a certain threshold are merged and considered (S. Henikoff & J.G. Henikoff,
1992). Therefore, different BLOSUM matrices are produced for each threshold. For example,
BLOSUM62 matrix (Fig. 10) is derived from merging several alignments with 62%, 80%, and
95% identity. This matrix is useful for scoring proteins that share less than 62% identity. By
increasing the clustering percentage, the ability of the resulting matrix to distinguish actual
from random alignments also increased. The numbers associated with BLOSUM matrices do
not have the same interpretation as those for PAM matrices. BLOSUM matrices with
smaller numbers represent more evolutionary distances while BLOSUM matrices with
higher numbers represent closer evolutionary distances. Consequently, BLOSUM matrices
are obtained based on entirely different type of sequence analysis and a much larger data set
than the Dayhoffs PAM matrices. The values of the BLOSUM scoring matrix are obtained
based on similar procedure applied to PAM matrices. However, BLOSUM scoring matrices
are calculated from 2 times the log base 2 of the odds ratio, as follows:


260

2
( , ) 2log
ab
b
b
M
S a b
P
| |
=
|
\ .
(10)
4.1.3 Comparison of the PAM and BLOSUM amino acid substitution matrices
Methods of computing PAM and BLOSUM scoring matrices have several important
differences. The PAM matrices are computed based on a mutational model of evolution
which assumes each amino acid change at a specific position is independent of the previous
changes at that position (based on Markov model).

Fig. 10. BLOSUM62 scoring matrix.
By predicting the phylogenetic tree of the studied sequences of each protein family, the early
changes that occur as protein diverge from a common ancestor during evolution is
identified. In order to derive matrices used for more distantly related proteins, short term
changes are extrapolated. In contrast, BLOSUM matrices are derived based on all observed
changes in an aligned region of a related family of proteins without considering the global
similarity between the considered protein sequences. Since these related proteins in the
family are known to be related biochemically, they should be derived from a common
ancestor. Generally, PAM model is designed to track the evolutionary origins of proteins but
the BLOSUM model is designed to find their conserved domains. Both use log-odd values in
their scoring systems (Vogt, 1995).


261
4.2 Nucleic acid scoring matrices
There are scoring matrices for DNA sequence alignments as amino acid scoring matrices.
A series of nucleic acid PAM matrices are calculated in similar way that amino acid PAM
scoring matrices are generated. To derive DNA PAM matrices first a PAM1 mutation
matrix which is representing 99% sequence conservation or 1% mutations across
evolutionary distance is calculated. It is upon the assumption that the frequencies of four
nucleotides in studied sequences are equal. Also all mutations from any nucleotide to any
other are equally likely. Thus, the four diagonal elements of the PAM1 matrix
representing no changes are equal to 0.99, but the other elements of the matrix
representing changes are 0.00333. For converting substitution matrix to scoring matrix just
as amino acid matrices, the values of the this matrix is used for producing log-odds
scoring matrices which is representing the frequency of substitutions expected to occur at
increasing evolutionary distances. For scoring DNA alignments with DNA PAM scoring
matrices, the lower numbered DNA PAM matrices is used for more alike DNA sequences
and the high numbered DNA PAM matrices are used for more diverge DNA sequences
along evolutionary distance (States et al., 1991).
5. Statistical analysis of alignments
One of the main challenges of the sequence similarity searches is to detect weather an
identified sequence similarity between DNA or protein sequences are statistically
significant. For two proteins that are quite similar and clearly grouped in the same family,
assessing the significance is not necessary. However, when we are dealing with two
sequences with no clear similarity, once the alignment is performed statistical analysis
becomes important. In such cases, biologists would like to know if the observed similarity
resulted from the alignment is obtained by chance or is authentic. A statistical test assists
biologists to identify the more distant related protein or DNA sequences from unrelated.
The assessing test is performed on the basis of the assumption that the alignment scores
follow a normal distribution. For evaluating the distribution of alignment scores, some
random sequences are generated by sequence shuffling technique. Analysis of the
alignment scores of random sequences reveal that the scores follow a type of normal
distribution called Gumbel extreme value distribution (Altschul & Gish, 1996; Altschul &
Bouguski, 1994). Generally, the statistical analysis of alignment scores for local alignments
is better understood than global alignments. Since, the Smith-Waterman algorithm reveals
regions of conserved or closely matching with a positive score, in random or unrelated
sequence alignments these regions are rarely found. Therefore, presence of such regions
in real sequences is significant while the probability of occurrence of such regions by
chance is close to zero. P-value is a suitable parameter which is used for identifying the
probability that a score of S or greater is obtained by chance between two unrelated
matched sequences of similar composition and length. Hence, very low p-value
corresponds to significant matches meaning that it is improbable the obtained score
occurred by chance. It is more probable that the high score occurred as a consequence of a
real biological or evolutionary relationship. However, a more common statistical
parameter which is reported by most softwares for quantifying the statistical significance
of an identified similarity is E-value or expected value. E-value is the expected frequency
of scores "S" occurred by chance. P and E values can be calculated for the two matched


262
sequences separately (calculating the probability of obtaining score between the two
sequences at least as high by chance) or for the database similarity searches. It must be
noted that, when the p-value for two matched sequences is low, E-values for searching a
large database can be quite large.
Assessing the statistical significance of a global alignment is very difficult, because
performing a global alignment using Needleman-Wunch algorithm and a suitable scoring
system produces many different alignments with quite similar scores. In aligning random or
unrelated sequences using global alignment method, the aligned sequences have very high
scores. Such investigations show that the tendency of global algorithm is to match as many
characters as possible. Regardless of this difficulty, a way is developed for assessing the
significance of a Needleman-Wunsch global alignment score. In this test the random or
unrelated sequences are created by shuffling the reference sequence(s) and the query
sequence(s) is aligned against random sequences in pairwise fashion, then the average of
scores of alignments is taken and is compared with the score of the real alignment in Z-score
parameter assuming that the overall distribution of the randomized score is normal:

x
z

o
= (11)
Where, x is the current score of two aligned sequences, p is the mean score of many
randomized sequence comparisons, and o is standard deviation of those measurements
obtained with random sequences. For evaluating the statistical significance of the two
aligned sequences the obtained Z-score is related to probability value. If all random
alignments have a score less than the authentic score, this indicates that the p-value is less
than 0.01 i.e. the probability of occurrence by chance is less than 0.01. As a result, the studied
sequences are significantly related.
Evaluation of statistical significance of local alignment scores of two sequences or a
sequence against a database of sequences (like BLAST and FASTA algorithms) is based on
E-value. In aligning sequences locally, the high scoring segment pairs (HSPs) are identified.
For BLAST algorithm, E-value is the most important statistics associated with BLAST output
describing the number of hits expected to occur by chance. Statistical evaluation of locally
aligned sequences is somewhat similar to that of global alignments, but the random
sequence alignment scores follow extreme value distribution which approximately
resembles a normal distribution with a positively skewed tail in the higher score range. The
goal is to evaluate the probability of obtaining a random alignment with a score equal or
higher than real sequences of interest. Thus, E-value is calculated as follows:

s
E Kmne

= (12)
Where, K is a constant value, m is the effective length of the query sequence, n is the
effective length of the random sequences, z is the scaling factor, and S is the score that
reflects the similarity of each pairwise comparison. The K and z parameters are described by
Karlin and Altschul (1990) and calculated by aligning 10000 random amino acid sequences
of variable lengths using Smith-Waterman method and a combination of the scoring matrix
and a suitable set of gap penalties for the matrix. Then values of the K and z were estimated
for each combination by fitting the data to the predicted extreme value distribution as
reported in Table 1.


263
Setting a threshold for E-value and p-value in database similarity searches, the sequence
similarities with scores lower than the threshold are considered significant. The sequences
with significant similarities are called hits. Based on the results of the search, the database
is grouped into two subsets called hits (positives) and non-hits (negatives). These subsets
conceptually grouped into true and false positives and true and false negatives. A true
positive is a hit enforced by a real biological pressure while a false positive is a hit without a
real biological relationship to the query sequence. A true negative is a non-hit with no
biological background to the query sequence and a false negative is also non-hit with a
biological relationship in reality.

Table 1. Statistical parameters (K, z) based on different scoring matrices and different
suitable affine gap penalties.
Evaluation of the results of a database search is performed by two complementary
measurements, known as sensitivity and specificity (Westhead et al., 2002). The sensitivity
(S
n
) is the proportion of the real biological relationships in the database that are detected as
hits and are calculated as follows:

( )
tp
n
tp fn
n
S
n n
=
+
(13)
where, n
tp
is the number of true positives, and n
]n
is the number of false negatives. The
specificity of the search is the proportion of hits corresponding to the real biological
relationships and is obtained as follows:

( )
tp
p
tp fp
n
S
n n
=
+
(14)


264
where, n
]p
is the number of false positives. To obtain more accurate results from database
searching, both sensitivity and specificity must be as close as possible to 1, but in practice
this is not possible. By increasing the threshold, the sensitivity is likely to increase (i.e.
obtaining more true positives and less false negatives), but the specificity is probably
decreased (i.e. more false positives). Hence, there is a trade off between these two quantities
for increasing the accuracy of the results. It should be noted that analysis of sensitivity and
specificity is only possible if the real biological relationships in the database is already
known and categories of true and false positives and negatives are created. The categories
are created from experimental determination of protein structure and function (Karlin et al.,
1991; Pearson, 1998).
6. Summary and conclusion
In this chapter, the sequence alignment methods and their basic concepts are described.
Alignment is the tool for inferring homology. Two types of sequence alignments including
global and local are studied that align a query sequence against a reference sequence. Both
methods guarantee to find an alignment with the highest scores based on the choice of
suitable scoring matrices. These matrices such as PAM and BLOSUM are computed based
on substitution matrices. Phylogeny is the core data upon which substitution matrices are
constructed. Evolutionary relationships between sequences come from the fact that species
undergo mutations over time. In mutated species amino acid sequences of proteins change
so that some residues are substituted by other biochemically similar residues. Substitution
matrices are built upon the close examination and quantitative analysis of mutations that
has been extensively described in this chapter. In order to align a query sequence against a
database the same basic concepts for two sequences (query vs. reference) are applied, but
faster algorithms are needed. The modified Smith-Waterman algorithms (BLAST and
FASTA) are presented for this purpose and are described in full detail. Ultimately, for
evaluating the statistical significance of the resulted alignments, these algorithms use
parameters such as P- and E-values. Using these values, real relationships are distinguished
from random relationships. The statistical analysis of alignment outputs are discussed in
detail in this chapter.
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13
Predicting Virus Evolution
Tom Burr
Los Alamos National Laboratory
USA
1. Introduction
Viruses are an important cause of human disease, often because they are highly
transmittable from human to human. A key tool from population genetics that can be
applied to the study of viruses is coalescent theory. Coalescent theory predicts genealogical
tree shapes as a function of how the studied organisms are evolving. Therefore, under its
model assumptions, coalescent theory can be used to infer aspects of the demographic
history of evolving organisms. For example, there are characteristics of tree shapes that
imply whether the organism population has been constant, growing, or shrinking in size
over time.
This chapter reviews some of the successes of coalescent theory in the context of inferring
aspects of virus evolution, using human immunodeficiency (HIV) and influenza viruses as
case studies. Next, the chapter describes limitations of coalescent theory, even as extended
to allow some forms of selection, population subdivision, and viral recombination. The
relatively new goal to predict influenza virus evolution (rather than infer past evolution) is
used to emphasize modeling needs beyond standard or extended coalescent theory models.
A new small-scale simulation that combines viral fitness with demographic population
structures such as family and work groups is then described as an example extension to
coalescent theory models.
Prediction goals include early detection of highly lethal new strains and improved vaccine
designs that anticipate future evolutionary directions. Regardless which evolutionary model
is used to predict virus evolution, because real virus evolution is complex beyond current
understanding, there will be substantial model error. Model error, model parameter
estimation error, and purely random effects can combine to make some forecast goals
unattainable. In these cases the most appropriate prediction is similar to what is often said
about stock markets: there will be change.
2. Background
Humans are susceptible to many viral pathogens, including the human immunodeficiency
virus (HIV) and the influenza virus. Although it is a relatively new goal in population genetics,
predicting virus evolution can help with vaccine design and with other mitigation strategies
(Bush et al., 1999; Ferguson and Anderson, 2002; Plotkin et al., 2002; Rambaut et al., 2008).
Using estimated phylogenetic (genealogical) tree shapes to infer aspects of evolution such as
organism growth rates has received far more attention to date (Felsenstein et al., 1999; Innan
and Stephan, 2000; Pybus et al., 2000; Stephens and Connelly, 2000; Ewing et al., 2004)


270
This chapter focuses on HIV and the influenza virus in the context of what might be
predicted about virus evolution. Influenza is a highly transmittable disease that infects
millions each year, resulting in many deaths. HIV is also transmittable through risky
behaviors and it too results in many deaths each year.
In population genetics, coalescent theory (Kingman, 1982; Stephens and Donnelly, 2000;
Burr et al., 2001; Ewing et al., 2004;) is a key tool that predicts genealogical tree shapes as a
function of how the studied organisms (taxa) are evolving. Therefore, under its model
assumptions, coalescent theory can be used to infer aspects of the demographic history of
evolving organisms. For example, there are characteristics of tree shapes that imply whether
the organism population has been constant, growing, or shrinking in size over time (Pybus
et al., 2000).
This chapter will first review some of the successes of coalescent theory in the context of
inferring aspects of virus evolution, using HIV ( Rodrigo et al., 1999; Burr et al., 2001;
Rambaut et al., 2001) and influenza viruses (Ferguson and Anderson, 2002; Plotkin et al.,
2002, Burr et al., 2002) as case studies. Next, the chapter describes limitations of coalescent
theory, even as extended to allow some forms of serial sampling, selection, population
subdivision, and viral recombination (Excoffier and Foll, 2011). The relatively new goal to
predict influenza virus evolution (rather than infer past evolution) is used to emphasize
modeling needs beyond standard or extended coalescent theory models. A new small-scale
simulation that combines viral fitness with demographic population structures such as
family and work groups is then described as an example extension to coalescent theory
models.
Most genetic data analyses rely on a forward model that specifies evolutionary forces and
associated probabilities describing how offspring are generated. Evolutionary forces
include drift, mutation, recombination, migration, and selection. Drift refers to random
change over successive generations due to finite population sizes. In the absences of
mutation and selection, the fraction of a population of size N having a given trait drifts
randomly somewhat like the number of heads in a set of N coin tosses. Mutations are
changes in the DNA sequence that occur for many reasons. Recombination (reassortment
in the case of influenza) refers to sections of genome that are broken and then recombined,
resulting in large genetic differences between offspring and parents, and complicating
phylogenetic analyses because different genome sections can have different genealogies.
Migration refers to exchange of genetic material among partly isolated subpopulations.
There are many other evolutionary forces, too many to describe here.
For simulating DNA sequences from a population, the state of art invokes coalescent theory,
which uses simplified models of the forward evolutionary process. These simplifications
allow inverse analytical solutions and corresponding simulation software, but with
questionable assumptions. This is done in order to avoid having to simulate directly from
the forward model and track the evolutionary histories. Sample genealogies can instead be
simulated by running time from the present toward the past and tracking probabilistically
when lineages coalesce to share a common ancestor. An example genealogy of a sample
taken at a single time from a population that is maintaining a constant population size is
given in Figure 1. These coalescent-based simulated sample units are then used to infer how
a population is evolving using features of the associated phylogenetic tree (Pybus et al.,
2000). In addition, analytical approximations used in inference invoke the same model
assumptions used in coalescent theory.


271
Agent-based models (Eubank et al., 2004) provide a richer framework than classical
epidemiology models of disease spread, such as the susceptible-infected-recovered (SIR)
model. Because agent-based models track individual rather than aggregate behavior, they
are believed to more reliably predict, for example, the impact of candidate mitigation
strategies such as vaccinations and isolation. In an analogous way, we describe predictions
for virus evolution that probably will require a higher-fidelity modeling framework than
coalescent theory and its extensions.
The following sections include HIV and influenza examples of using coalescent theory to infer
aspects of prior evolution, limitations of coalescent theory to infer future HIV and influenza
evolution, introduces the new small-scale simulation that combines viral fitness with
demographic features, and discusses limitations of our current ability to predict viral evolution.

Fig. 1. The most recent common ancestor and sample genealogy from an evolving
population. At time t a sample is collected, and at time 0 in the past, all individuals in the
sample coalesce to share a common ancestor. For simplicity here, the population size is
assumed to be constant over time, with each individual at time 0 represented by a dot (not
all individuals are shown).
3. HIV and influenza examples
3.1 Example 1: HIV
Within host. A coalescent model within individuals has been applied (Rodrigo et al., 1999)
to analyze HIV-1 viral load data from infected individuals after the administration of an
HIV-1 inhibitor to estimate the HIV generation time in vivo. The estimate was 1-2 days,
which agreed well with an estimate based on a different approach (Perelson et al, 1996),
although it assumed nonrecombining DNA sequences from a population of constant
effective size N. Consider samples from two sample times, separated by d days. The number
of days per generation is estimated as dn(n-c)/2Nc where c is the number of coalescent events
that have occurred, d is the number of days between samples. The method assumed: (a) the


272
population size N is constant; (b) the estimated phylogeny is the same as the true genealogy
of the sampled individuals, and (c) the exchangeability assumption that each individual
virus has the same propensity to reproduce. Implicit in (b) is the further assumption that
recombination, migration, and selection do not interfere with the ability to estimate the true
phylogeny. Further, an approximate technique for accommodating serial samples is
required, which has recently become available (Excoffier and Foll, 2011).
Between host. An example (Burr et al., 2001) involves whether the 8 to 10 approximately
equidistant subtypes of HIV-1 (type M) could have arisen under available models of how
HIV is evolving (Fig. 1). To examine this, coalescent theory was used to simulate DNA data
from a very simplified forward model of how HIV is evolving at both the macro and micro
levels (see Section 4.1). This provided a reference distribution against which to compare the
data. If features of the observed data (such as the ratio of the between-subtype to within-
subtype genetic distance) are in the tail of the coalescent-theory-based reference distribution
of those same features, then the forward model used to simulate the data is not credible.
Examples of phylogenetic trees estimated from coalescent-based simulated data are given in
the right box of four subplots in Figure 2. Notice that subtypes are expected to arise in
examples (b), (c), and (d), but not in (a). Subplot (a) is the classic star phylogeny that arises
when the underlying population size is growing rapidly, forcing most coalescent events to
occur early in the growth period, and all at nearly the same time.

Simulated data: 4 macro growth rates
(c) N = N
0
, then N= N
0
e
rt
(a) N = N
0
e
rt (b) N = N
0
(d) N is quadratic
from1970 to 1990

Fig. 2. HIV, env region. Consensus trees (of 100 bootstrap samples) using maximum
likelihood for real (the left plot) HIV (env gene) sequences and for coalescent-based
simulated (the four right plots) sequences under different assumptions about the time
behavior of the number of infecteds N.
3.2 Example 2: Influenza
Figure 3 shows a principal coordinate (PC) plot (Venables and Ripley, 1999) of a 129-by-129
distance matrix based on the nucleoprotein (NP) region of 129 influenza viruses isolated
Real HIV (env gene)


273
from humans, swine, and avian hosts. PCs provide a low dimensional way to represent a
distance matrix. For this data, all pairs of distances can be quite accurately reproduced using
only the first two PCs as in Figure 3. It is known that the NP region maintains a type of
species signature such as depicted in Figure 3 (Burr et al., 1999, 2002; Chen et al., 2006). A
key aspect of influenza evolution is the fact that avian and swine hosts occasionally act as
reassortment vessels for human influenza, resulting in dramatically different strains that
evade effective human immune response. As an aside, the term reassortment seems to be
applied only to influenza, presumably because its genome consists of eight distinct
segments. For our purposes, reassortment is the same as recombination, in which sections
of the genome get recombined (Forrest and Webster, 2010).
Figure 4 shows a PC plot of a distance matrix based on the hamagglutinin (HA) region of
influenza viruses. Figure 5 is a phylogenetic tree built using neighbor joining (Swofford et
al., 2000) of the same HA sequences.
Figures 4 and 5 illustrate (Nelson and Holmes, 2007) that the HA region appears to display
the effects of positive selection due to the cactus-like structure with most lineages dying out.
This cactus shape is unlike the classic star-like shape HIV trees of type M as in Figure 2.
Such a cactus shape can also arise without positive selection from a combination of serially
sampled taxa and sequential random population bottlenecks (which can occur in influenza
due to its strong seasonality). Therefore, the cactus shape by itself can indicate but does not
prove that positive selection is in effect. More formally, the statistical notion of identifiability
probably does not hold in this context. Model identifiability implies that as sample size
increases toward infinity, model parameters can be uniquely estimated (see Section 6).

Fig. 3. Principal coordinate plot of the evolutionary distances among 129 influenza viruses
extracted from human (H), avian (A), and swine (S) hosts. Distances are computed for the
Nucleoprotein region of the virus, which exhibits species signatures. Among the 129, there
are 14 misidentified taxa. However,the human (H) that is clustered with the avian group
was known to have been infected by poultry. There are 44 avian, 57 humans, and 28 swine,
all available from www.flu.lanl.gov.


274

Fig. 4. Principal coordinate plot of the influenza viruses (HA region) found in humans. Digit
= year, Black = 1960s, Red = 1980s, Green = 1990s. Genetic drift and strain extinctions are
known to occur (cactus shape of typical tree).

Fig. 5. Neighbor joining tree of the same HA sequences in humans that was used in Fig. 3.


275

Fig. 6. Genetic distance versus time (top) and versus difference in space (bottom) for the HA
region.
4. Limitations of coalescent theory for predicting HIV and influenza evolution
Coalescent theory leads to tremendous insights and powerful simulation and inference
tools. However, limitations of coalescent techniques include (a)-(d) as follows:
a. Little is known concerning accuracy and robustness of coalescent theorys restrictive
assumptions in many settings, although some forward models are known not to be well
approximated by any coalescent model (depending on the relative time scales of
various evolutionary effects such as drift, migration, and selection) (Sjodin et al., 2005);
b. Inference methods (Pybus et al., 2000; Stephens and Donnelly, 2000) invoke coalescent
approximations to estimate the probability of candidate branching orders as part of the
inference process. This leads to the undesirable situation of forcing a zero mismatch
between the inference methods assumptions and the assumptions regarding how the
population is evolving;
c. Coalescent theory is expanding along with associated software for implementation, but
no current coalescent-based software includes all extensions to the original coalescent
theory. However, one new option (Excoffier and Foll, 2011) for coalescent-based
software includes many of the standard evolutionary features such as serial sampling,
recombination, and geographic isolation;
d. Building trees supports inferences regarding, for example, whether a virus strain
appears to be a natural branch from historical strains, or whether the strain seems to
have made an unnatural leap indicating bioengineering. However, key coalescent
assumptions that are violated by both HIV and influenza viruses are that all subtypes
are equally transmissible and there is no recombination. Therefore, although to a
limited extent and under restrictive assumptions, extensions to coalescent theory have
Genetic distance
Difference in time (yrs)
Genetic distance
Difference in space (arbitrary units)


276
been made to accommodate recombination, selection, overlapping generations, and
population subdivision, there are cases where the theory is either inadequate or the
sensitivity of its conclusions to its assumptions is unknown. The corresponding
inference quality using estimated trees is also unknown; the state of the art is therefore
to quantify precision, but not accuracy.
The forward model is a key component of total uncertainty associated with population
genetics inferences. The current approach is: specify an amenable-to-coalescent-theory
forward model for how a population is evolving that includes for example, population size,
structure, and selection effects; identify the coalescent effective population size N
e
(Sjodin et
al., 2005) in the nearest available coalescent model, which is often a complicated task. Then,
use the closest coalescent model to simulate sample genealogies under restrictive
assumptions about the population and the sampling process. The N
effective
notion arose from
coalescent theory by mapping the actual population size N in a population that violates
some coalescent assumptions (such as nonoverlapping generations) to a different size
N
effective
such that in some aspects, the actual and model populations evolve probabilistically
in approximately the same manner. Coalescent theory was originally applied to macroscopic
populations such as plants and animals (for example, Innan and Stephan, 2000); it has also
been applied to microscopic populations such as DNA sequences from virus populations
(for example, Rodrigo et al, 1999).
Coalescent theory will continue to provide insight into evolutionary processes; however, it
is currently unknown how robust associated inferences are with respect to model violations.
For example, Innan and Stephan [5] assumed the wild plant Arabidopsis thaliana (useful for
genetic studies because of its well known demographic history and genome) consists of
many isolated colonies, each having negligible genetic variation within a colony. They
applied a coalescent model (correcting for the growing population size of A. thalina) to
simulate the probability distribution of Tajima's D statistic against which to compare the
observed D in real samples, as a test for selection. Tajima's D statistic is based on the
difference between two estimates of the amount of variation (one using the number of sites
having genetic variation and the other based on pairwisedifferences between individuals).
They concluded that there was evidence for selection (distinguishing the type of selection,
such as balancing or purifying is a separate challenge). Because of the simplifications
inherent in the coalescent approach, it is currently unknown how robust the evidence for
selection is in this for A. thalina.
4.1 Example 1: HIV
Coalescent models of HIV reproduction within an individual might be adequate (Rodrigo et
al., 1999); however, these would become prohibitively unwieldy if all HIV-infected humans
were modeled. For example, all models must specify the macro components such as the
reproductive rate in susceptible populations and/or subpopulations.
As mentioned in Section 3.1, an investigation into the development of the HIV subtypes led
to an application of coalescent theory to model the population dynamics of HIV (Burr et al.,
2001). Figure 2 illustrates the approach taken. Various features (such as the ratio of the
between-subtype to within-subtype genetic distance) involving the subtypes of real HIV
sequences (env gene) were compared to the same features in corresponding coalescent-
based simulated data. However, it became apparent that it would be necessary to
implement a model that made less restrictive assumptions than coalescent theory (Burr et
al., 2001).


277
One possible new way to simulate sequences is to track each HIV case by geographic region
including all known transmission routes such as sex, needles, blood transfusions, and
mother-to-child, and track the genealogy of each case. One would then sample ~100
simulated sequences from around the world or in specified regions at a snapshot in time, or
distributed in time, and distributed spatially in either case. With careful bookkeeping one
could deduce the sample genealogy (which 2 samples coalesced first to their most recent
common ancestor (MRCA), which samples coalesced next, etc.) back in time until all 100
sequences coalesced to the single MRCA. This would produce 99 coalescent times and
sample identities, which define the genealogy of the sample. This genealogy could also be
thought of as the true evolutionary tree for the sample to be compared to coalescent-based
genealogies.
Related to the origin of the HIV subtypes is the goal to predict the stability of the subtypes
because current vaccine design approaches rely on mosaic pseudo-HIV viruses that
exploit the known characteristic or representative sequence of each subtype (Barouch et al.,
2010). Although a few new subtypes have been defined since the original, the M clade trees
with 8-10 subtypes have been remarkably stable over time (Korber and Myers, 1992; Burr et
al., 2001). Both within-host and between-host modeling efforts should allow for multiple
viral sequence types within hosts, because within-host variation in contemporaneous HIV
sequences isolated from various regions of the genome exhibit substantial variation, easily
up to 10% differences.
4.2 Example 2: Influenza
Imagine a particularly bad flu season. Not only does it appear that more people are infected
than normal by early November, but there are anomalous deaths. Could this be a bio attack
or perhaps a another human-to-human transmittable version of the swine-origin influenza
A (H1N1) virus?
As Figure 6 suggests, there is empirical evidence that a time gap of three or more years is
sufficient for a temporal signature. For example, strains isolated in 1993 should be
genetically distinct from strains in 1996 or later (Burr et al., 1999). Therefore, we might be
suspicious in 2012 if the strain looks like a 2008 strain. However, the empirical evidence
assumes a constant population size because the genetic distance between two samples
depends on the coalescent time since they evolved from the same ancestral sequence. And
the time since the two samples shared a common ancestor depends on several factors,
including how the population is structured and the size and growth rate of the population.
If some of these factors change dramatically, then the three-year rule would become either
shorter or longer. Currently, coalescent methods either hold these factors constant over time,
or extensions to the approximations have not been implemented. Therefore, empirical
reconstructions of phylogenetic trees such as those in Burr et al. (1999) are incomplete for
assessing the robustness of candidate signatures. The corresponding inferences thus have
unknown reliability.
The N
effective
concept is part of the success of coalescent theory, including in the influenza
context Bedford et al (2010) estimate N
effective
for influenza A using a coalescent model that
includes subdivision and migration. Bayesian Evolutionary Analysis Sampling Trees
(BEAST, Pybus et al., 2007) was used to estimate assuming both N and are constant over
time. In this example, it could be important that observed and reported influenza mutation
rates need not be stable over time for several reasons, including the fact that N
effective

changes with time.


278
In influenza, genome reassortment, selection, the presence of multiple strains and multiple
hosts, and host immunity all complicate matters. Given what is known about influenza
evolution, what might we predict today about influenza evolution? Two related prediction
goals to consider for influenza are: (1) in a given year, predict which new strains are most
likely to be in the surviving lineage, and (2) predict the prevalent strains in the next year, so
that vaccine design can be most effective.
Concerning prediction goal (1), Bush et al (1999) proposed a prediction method that
involved whether influenza isolates on lineages having the most changes in positively
selected codes were more fit that other isolates. At least 18 of the 329 AA codons in H3
HA1 are thought to experience positive selection, with mutations favoring new variants that
can escape host immunity. An AA sequence was defined as more fit if it is more closely
related to surviving lineages than another contemporary strain.
Concerning prediction goal (2) the world health organization (WHO) recommends three
strains to target in the vaccine for each flu season. Plotkin et al. (2002) use non-hierarchical
clustering over time to evaluate the number of HA1 sequences within each cluster over time.
This leads to a sequence-based algorithm to choose vaccine strains and the recommended
strains differed from the WHO recommendation in 9 of 16 years in the study period from
1985 to 2000. A limitation of the Plotkin et al (2002) study is the biased sampling used by
WHO in which novel strains are deliberately overrepresented in the database.
The new small-scaled agent-based simulation described in Section 5 addresses both
prediction goals 1 and 2.
5. New small-scale agent-based simulation for influenza
In choosing/developing an evolutionary model it is of course important to consider the
modeling goals. What are the prediction goals? How should the dynamic host/pathogen
system be modeled? Which hosts should be included? Human, swine, avian, other? Is it
sufficient to use a detailed model of a region such as New York state and a less detailed
model of the outside region?
The basic susceptible-infected-recovered (SIR) model in classical epidemiology
mathematically describes average population behavior using differential equations to move
from S to I to R. This SIR model has been extended in various ways including structures
such as contact groups and stochastic effects such as varying contact rates (Burr and
Chowell, 2008,2009). Figure 7 gives examples of different simulated outbreak shapes in a
small population of 1000 individuals. The number of newly infected is plotted each day for
the simulated data. The small population is either (a) an unstructured population with all
individuals equally in contact with all other individuals; (b) a randomly generated network
model in which individuals are only exposed to member of their own clique, but some
individuals belong to multiple cliques. (c) A network model with cliques assigned to nodes
in a lattice; (d) a more realistic spatial network in which cliques belong to small geographic
regions. A clique is a small group of individuals for which the contact probability is
relatively high and assumed to be the same between each member of the clique. Various
signatures of non-homogeneity using the shape of typical outbreak curves were developed
for such network models, which were then shown to be detectably different from outbreak
curves from the basic SIR model with homogeneous individuals all equally mutually
exposed (Burr and Chowell, 2008). One concept that arose in Burr and Chowell (2008) is that
predictions of the total number of infected based on the basis SIR model were often quite


279
wrong in the structured population. This is an example of using failed predictions to
determine that more fidelity is needed than the SIR model provides.
There has been progress toward merging SIR models with viral fitness. Minayev and
Ferguson (2009a, 2009b) extended SIR models by including two key notions: cross immunity
to similar strains in a host that has been previously infected by a similar strain, and transient
strain-transcending immunity.
At least two related empirical studies have been published, both previously mentioned
(Bush et al., 1999; Plotkin et al, 2002) In Bush et al. (1999), the number of AA changes in a
lineage appeared to convey a selective advantage in the following sense. The lineage for
which the most AA changes occurred were more likely to be represented in the surviving
lineages. That is, mutation conveys selective advantage, which is believed to be the case
simply because the human host has some immunity to prior strains. At this point, strain
will be defined following Plotkin et al. (2002) as arising from cluster analyses bases on the
Manhattan metric that counts the number of AA differences between pairs of sequences.
With that definition of a strain, and using 2 AA changes as the threshold above which a new
strain is defined, Plotkin et al. (2002) reported empirical assessment of the number of strains
by calendar year in influenza samples.
5.1 Evidence of departures from standard models
Graves and Picard (1999) report evidence of violations of the classic SIR model for influenza.
Signatures of departure from SIR (Burr et al., 2006) characterize departures from the one
season fits all assumption (which assumes each flu season occurs like clockwork, peaking
in the winter during the same weeks, etc) using a hierarchical model that captures year-to-
year variation in baseline, and peak onset and duration (Burr et al., 2006). Burr and Chowell
(2008) use a reference distribution of simulated outbreak curve shapes to assess whether a
collection of simulated and real outbreak curves follow SIR-type models. On that basis,
many real outbreaks do not follow SIR-type models.
5.2 Description of the new small-scale simulation
In the context of predicting viral evolution as considered here, the SIR-type model must be
extended include population demographics and characteristics of the virus. Minayev and
Ferguson (2009a,b) develop one approach to include viral characteristics. Our approach to
be described in this section is similar, but is entirely stochastic and allows for demographic
structure. With the present implementation, population sizes of approximately 10,000 can
complete in reasonable (tens of minutes) run times, so the simulation is small-scale.
Here is pseudo code to describe the new simulation. In some cases, parameter names such
as average.duration.of.infection are used to clarify.
Pseudo-simulation code (Example R (R, 2004) code named flu1( ))
1. Initialize the population matrix and the matrix of AA sequence.
pop.matrix is N rows (individuals) and 30 columns with:
Column 1 is current age.
Column 2 is infection status (0 = susceptible, 1 = infected, 2 = recovered and not susceptible).
Column 3 is the number of times the individual has been infected.
Column 4 is the family group. Column 5 is the work group. Column 6 is the "other group."
Column 7 is the time of first infection. Column 8 is an integer denoting the AA sequence of
infection 1. Column 9 is the donor ID for infection 1.


280
(a) Unstructured population, SIR model
Day
20 40 60 80 100
0
5
0
1
0
0
1
5
0
(b) Random network
Day
20 40 60 80 100
0
5
0
1
0
0
1
5
0
2
0
0
(c) Lattice network
Day
20 40 60 80 100
0
5
0
1
0
0
1
5
0
(d) Spatial network
Day
20 40 60 80 100
0
1
0
0
3
0
0
5
0
0
No. of newly infected individuals each day

Fig. 7. Example time series of newly infected individuals in simulated SIR models of
populations of 1000 individuals. (a) basic SIR model; (b) a randomly generated network model
in which individuals are only exposed to member of their own clique, but some individuals
belong to multiple cliques. (c) A network model with cliques assigned to nodes in a lattice; (d)
a more realistic spatial network in which cliques belong to small geographic regions.
Columns 10-12 are the same as columns 7-9, but for the second infection by the individual.
A maximum of 30 infections is allowed and then each new infection is recorded in the last 3
columns by writing over the previous data.
AA. seq.matrix begins with 2 rows (by default) and 329 AA sites (columns).
The default is to being with two random but distinct AA sequences.
At each time step (the default time step is 1 day), any of several events can occur:
2. There is a probability for each individual to change status from S (0) to I (1), or from I
(1) to R (2), or from R back to S.
Any susceptible (S) individuals that an infect (I) individual contacts in the respective family,
work, and other groups leads to a probability of infection determined by two parameters.
First, there is a force of infection parameter for each of the three group types that
characterizes how strongly individuals in the three group types interact. Second, the
similarity of the infected individual's current strain to the closest strain of a given
susceptible is computed and the cross-immunity function is calculated.
The value of the function in Minayev and Ferguson (2009b) alters the transmission
probability accordingly. Cross immunity modeled by decreases to zero as a smooth


281
function of time (examples given below), with an average of 10 year total immunity from
identical strains. And, values of .a and .b in can be altered to decease or increase the
degree of cross-immunity as a function of the Manhattan distance between strains. The
cross-immunity concept is that S individuals who have had the strain of the potential donor
I are less susceptible to infection. As Plotkin et al (2002) describe, ideally a distance measure
between two sequences should somehow reflect immuniological properties of the
corresponding viral proteins. Although steps have been taken in that direction (Lapedes and
Farber, 2001), more research is required before similar metrics can be defensibly applied in
modeling contexts such as our new small-scale simulation.
Any newly infected host will have the donor strain, but the simulation allows for mutation
to a new strain. There are 329 H3 HA1 (Bush et al., 1999) amino acid (AA) sites with one
estimate of the effective mutation rate N being 0.0057 nucleotide substitutions per site per
year. Of the 329 AA sites, at least 18 have exhibited positive selection effects (Plotkin et al.,
2002). Here we will not consider estimation error in N, so the simulation default value is
N = 0.0057 x 3 = 0.171 per AA site per year. A technical issue arises here because we use the
actual popluation size N rather than the effective size N
effective
. It would be more appropriate
to use N
effective
, but that value is currently unknown in the context of this model population.
In future work, N
effective
could be defined and estimated on the basis of the number of
observed distinct sequences during outbreak.
If a newly infected incurs any mutations, add the new strain to AA.seq.matrix, increasing
the number of rows by one. Columns in pop.matrix identify which strains each host has had
in the sequence matrix of all strains ever experienced in the model population
3. Any infected can recover.
The per-step recovery probability is 1/(average.duration .of. infection). The time to recovery
is therefore a geometric random variable with average duration
average.duration.of.infection.
4. Any recovered can lose immunity.
The time t from recovery to immunity is random, with t ~ Normal(avg.time.to.immunity,).
The default simulation values are
avg.time.to.immunity=10 years and = 1 year.
If at any time step (day) the number of infected is 0, then the infection would die out.
Therefore, a reintroduction of new infected occurs at a random time with a user-specified
average value with default value of 1 year, representing the typical time gap between outbreaks.
Figure 8 plots the percent currently infected at each time step for one 7-year realization of
10,000 individuals. A key output of flu1 is the current strain of each infected individual at
each time step. This allows us to consider strategies in Bush et al (1999) and in Plotkin et al.
(2002) for prediction goals (1) and (2) described earlier in this section. Figure 9 plots four of
the eight strains that emerged during the 7 simulated years. Following Plotkin et al. (2002),
sequences were regarded as being the same strain if the number of AA differences among
the 18 positively-selected AA sites is 2 or less. Equivalently, sequences were regarded as
being distinct strains if the number of AA differences is 3 or more. Figures 8 and 9 used the
values .a = 0.4, .b = 0.95.
Experimentation with flu1 to generate multiple realizations of outbreaks having identical
parameter values allows us to examine the role of chance in our models. Experimentation
with flu1 with different parameter values allows us to examine the effects of parameter
changes. Small numerical experiments with flu1 to date has lead to the following
following conclusions:


282
1. The .a and .b in are as critical to the size of each outbreak as the overall transmission
probabilities within the family, group, and other groups. For example, the function
takes values 0.990, 0.891, 0.792, 0.693, 0.594, 0.495, 0.396, 0.297, 0.198, 0.099, 0.000,
for distances of 1, 2, , 11, respectively, for .a = 0.1, .b = 0.99, and takes values 0.8,
0.4, and 0.0, for distances of 1, 2, 3 , respectively for .a = 0.5, .b = 0.8. The modeled
transmission probability is multiplied by 1- so for .a = 0.1, .b = 0.99 there is very little
chance of a susceptible individual acquiring influenza from a host having an AA
sequence that differs in only 1 position among 18 positions from a strain the susceptible
has had within the duration of immunity (10 years on average for example). This
means that a single AA mutation can have dramatically different effects on
transmission probability depending on .a and .b. Qualitatively, this is anticipated
because if immunity to a new strain is very high in individuals with previous infection
by a similar older strain, then the average outbreak size will be small if previous
outbreaks due to the older strain were large.
2. The values of .a and .b in are also critical to the typical number of strains maintained
in the population and to whether change occurrence of a large number of mutations in a
newly infected will have strong selective advantage by avoiding the collective immune
experiences of available human hosts. It is currently unknown whether the observed
number of strains could adequately provide a model such as flu1 with estimates of .a
and .b (See section 6).
3. As expected, the group structures can lead to outbreak shapes that differ from classic
SIR outbreak shapes (Burr and Chowell, 2008). This is evident in comparing the
outbreak shapes in Figure 8 to the SIR-model outbreak in Figure 7a for example. The
shapes in Figure 8 fall off very sharply, more like the spatial network in Figure 7d.
4. In population genetics, the composite parameter
effective
2N where is the
mutation rate determines the rate of genetic changes and the expected amount of
diversity in a random sample (see Section 6). The
effective
N concept for influenza
sequences was addressed in Bedford et al. (2010), but as mentioned in Section 4,
observed genetic diversity is interpreted in the context of idealized evolutionary models
that are amenable to coalescent theory. More experiments with flu1 are planned, and
if possible, an approximate coalescent model as implemented in available software will
be applied so that the adequacy of coalescent-based approximations can be evaluated in
the context of simulated flu outbreaks. We caution that many genetic effects of
influenza evolution are omitted from flu1 and from any available coalescent-based
simulation.
6. Model Identifiability and Inference
Model identifiability is a key statistical concept. A model is identifiable if its parameters can
be accurately and precisely estimated as the sample size increases toward infinity. In
population genetics, a key parameter that arises from coalescent theory considerations is the
composite parameter

effective
2N , which determines the rate of genetic changes. Many
studies address methods to estimate but because
effective
N and the mutation rate enter
as a product, they are confounded, leading to a lack of identifiability unless auxiliary data
is used to separately estimate
effective
N or and a strict evolutionary clock is assumed,
meaning that is constant over time and lineages.


283

Fig. 8. Example simulated outbreaks from flu1. The number currently infected is plotted by
day for years.
For a particular evolutionary model, inference is possible using Bayesian evolutionary
analysis, for example, by using BEAST (Pybus et al., 2000) that relies on Markov Chain
Monte Carlo, resulting in a posterior distribution on model parameters. The Bayesian
approach allows one to repeatedly sample from the posterior probability of model
parameters, and then generate hypothetical future genetic data for each set of model
parameter values. This approach provides an envelope of possible future multivariate time
series of genetic data from each sampled subject. It is computationally challenging even for a
given model of evolution.
In Burr and Chowell (2008), in simulated data from models with demographic structure but
no host immunity or viral strain information, predictions from SIR models with parameters
estimated from the early portion of an outbreak were often badly wrong. Such bad
prediction errors can indicate model violations, perhaps eventually leading to more
appropriate models. To our knowledge, using prediction quality to assess model adequacy
in this context is new. However it is possible that multiple wrong models provide adequate
predictions, so model identifiability remains a research topic in this area.


284

Fig. 9. Percent infected by day with strain 1, 2, 3 or 4 for the same simulated 7 years as in
Figure 8.
7. Conclusions/summary
Coalescent theory and its success in some contexts at inferring aspects of past virus
evolution were described. Then the argument was made that relatively new goals to predict
aspects of virus evolution will require higher fidelity modeling that is anticipated to be
available via coalescent theory or its extensions.
As a step toward high fidelity modeling, a small-scale agent based simulation was described
and example results presented. For influenza, two prediction goals were considered: (1) in a
given year, predict which new strains are most likely to be in the surviving lineage, and (2)
predict the prevalent strains in the next year, so that vaccine design can be most effective.
The new small-scale simulation code flu1in R can provide insight into the feasibility of
meeting these goals, but it too makes restrictive modeling assumptions with unknown
accuracy. A related concept was described that involves using prediction quality on
simulated data that follows an assumed model to assess whether prediction performance on
corresponding real data indicates model violations. Model violations that are evident from
poor prediction quality can help prioritize future upgrades to the models.


285
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Molecular Systematics, Edited by Hillis, D.; Moritz. C., & Mable, B, SInaer,
Sunderland, Mass. 1996.
Stephens, M., & Donnelly P. (2000). Inference in Molecular Population Genetics. J. Royal
Statistical Soc B 62(4); 605-655.
Venables, W., & Ripley, B. (1999) Modern Applied Statistics with Splus, Springer: New York.
Part 5
Protein Structure Analysis

14
A Bioinformatical Approach to Study the
Endosomal Sorting Complex Required for
Transport (ESCRT) Machinery in Protozoan
Parasites: The Entamoeba histolytica Case
Israel Lpez-Reyes
1
, Cecilia Bauelos
1
,
Abigail Betanzos
2
and Esther Orozco
2,3

1
Instituto de Ciencia y Tecnologa del Distrito Federal,
2
Centro de Investigacin y de Estudios Avanzados del Instituto Politcnico Nacional,
3
Universidad Autnoma de la Ciudad de Mxico,
Mxico
1. Introduction
1.1 The potential of bioinformatics for the study of protein structure and function
Proteins are macromolecules formed by amino acid polymers that regulate cellular
functions. Each protein is composed by the repetition and combination of 20 different amino
acids, whose order is determined by the genetic code. To perform their biological functions,
proteins fold into one or more specific spatial conformations, determined by non-covalent
interactions such as hydrogen bonding, ionic interactions, Van der Waals forces and
hydrophobic packing, and covalent interactions, such as disulfide bonds (Chiang et al.,
2007).
Determining the structure and function of a protein is a milestone of many aspects of
modern biology to understand its role in cell physiology. Bioinformatics is the research,
development or application of computational approaches for expanding the use of
biological, medical, behavioral or health-related data. It also includes those tools to acquire,
store, organize, archive, analyze or visualize infomation. Over the past years,
bioinformatical tools have been widely used for the prediction and study of protein biology.
Moreover, bioinformatical tools have revealed the existence of protein interactomes,
demonstrating the interaction among distinct biomolecules (protein-protein, protein-lipids,
protein-carbohydrates, etc.) to perform cellular processes (Kuchaiev & Przulj, 2011).
During the last decades, genome sequencing projects together with bioinformatics programs
and algorithms have enormously contributed to understand protein structure, protein
interactions and protein functions. At present, over six million unique protein sequences
have been deposited in public databases, and this number is increasing rapidly. Meanwhile,
despite the progress of high-throughput structural genomics initiatives, just over 50,000
protein structures have been experimentally determined (Kelley & Sterberg, 2009). The
greatest challenge the molecular biology community is facing today is to analyze the wealth
of data that has been produced by the genome sequencing projects, where bioinformatics


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has been fundamental. Traditionally, molecular biology research has been carried out
entirely at the laboratory bench, but the huge increase in the amount of data has made
necessary to incorporate computers and sophisticated software into research.
Additionally, availability of genome databases for distinct organisms has improved our
knowledge on the way to elucidate the last universal common ancestor. In conclusion,
analyzing and comparing the genetic material of different species is an increasingly
important approach for studying the numbers, locations, biochemical functions and
evolution of genes and proteins.
In this review, we selected a particular scientific case to emphasize the usefulness and
potential of bioinformatics in addressing a biological problem.
Most cellular processes use scaffold proteins to recruit other proteins and to facilitate their
correct interaction and functioning. Thus, we focused on the very little studied scaffold
proteins that form the Endosomal Sorting Complexes Required for Transport (ESCRT)
machinery during protozoan endocytosis, a fundamental process for cell survival. Here, as a
study case, we aimed to highlight the possible identity, function and interactions of ESCRT
complexes in Entamoeba histolytica, as determined by the use of bioinformatical tools.
1.2 Role of the ESCRT in endocytosis
Endocytosis is a crucial process in multiple cellular and physiological events, including
nutrient uptake, virus budding, cell surface receptor downregulation and cell signaling. It
involves the internalization of molecules or particles of different sizes from the external
environment, through membrane remodeling and vesicle formation events (de Souza et al.,
2009). In endocytosis, a huge number of interactomes are involved. In the study of the
highly complex endocytosis process, bioinformatics databases and computational tools have
been of enormous value.
Several plasma membrane proteins interact with target molecules (cargo) to internalize
and transport them along the endocytic pathway. Depending on their function, membrane
proteins are recycled back to the cell surface or degraded at lysosomal compartments
together with cargo. Delivery of endocytosed cargo for degradation occurs through the
fusion of intracellular vesicles called early and late endosomes that finally reach
lysosomes.
In the majority of cell types, late endosomes fuse among them to form multivesicular bodies
(MVB), which are essential intermediates for nutrient, ligand and receptor trafficking
(Williams & Urb, 2007). The best characterized signal for entering cargo molecules into the
degradative MVB pathway is ubiquitination. Ubiquitination is a conjugation event in which
a highly conserved 76 amino acid protein called ubiquitin, is covalently attached for cargo
labeling. Most of the cargo proteins that accumulate in MVB are marked by a single
ubiquitin, which is recognized by a specific and conserved protein machinery termed
Endosomal Sorting Complex Required for Transport (ESCRT) and whose function is
fundamental during endocytosis (Williams & Urb, 2007).
The ESCRT machinery was first characterized in yeast. It consists of a group of vacuolar
protein sorting factors (some of them called Vps), which form different multimeric
complexes (ESCRT-0, -I, -II and -III) that bind among them but also associate to accesory
proteins and endosomal membrane lipids to perform the whole endocytic process (Fig. 1)
(Hurley & Emr, 2006).
A Bioinformatical Approach to Study the Endosomal Sorting Complex Required for
Transport (ESCRT) Machinery in Protozoan Parasites: The Entamoeba Histolytica Case

291

Fig. 1. The ESCRT machinery involved in the endosomal MVB pathway.


292
(A) Eukaryotic cells internalize cargo molecules from the external environment by endocytic
processes. These molecules transit along several compartments for surface recycling or
degradation. The degradation pathway involves an endomembrane system constituted of
membrane bound organelles called endosomes that mature from early to late
endosomes/MVB for finally cargo delivering into lysosomes. According to (B), at late
endosome level, molecules to be incorporated into the degradation pathway should be tagged
with ubiquitin. In yeast, ubiquitination of cargo proteins is mediated by the ubiquitin ligase
Rsp5 and by Bul1. Then, the ESCRT-0 complex initiates the MVB sorting process by endosomal
membrane binding through the Vps27 domain, and ubiquitin recognition of cargo by UIM
domains present in Vps27 and Hse1 proteins. Subsequently, Vps27 activates the ESCRT-I
complex through its interaction with Vps23. Ubiquitinated cargo is recognized by ESCRT-I
(via the UEV domain of Vps23) and by ESCRT-II (via the NZF domain of Vps36). Vps36 has an
extensive positively charged region with high affinity to phosphoinositides, allowing ESCRT-II
attachment to endosomal membranes. Then, ESCRT-III concentrates cargo proteins into MVB.
ESCRT-III also associates to accessory proteins such as Bro1 and Doa4. Importantly, the Vps4
ATPase catalyzes the dissociation of ESCRT complexes to initiate new cycles of cargo sorting
and transport. Together, ESCRT-0 to -III and -accessory proteins direct cargo sorting, vesicle
fusion, and MVB biogenesis (modified from Hurley & Emr, 2006).
Cargo ubiquitination is mediated by the Rsp5 ubiquitin ligase and Bul1 protein. Then, cargo
sorting through the MVB pathway initiates with the association of Vps27 and Hse1 proteins
to make up the ESCRT-0 complex. Vps27 has a FYVE (Fab1, YOTB, Vac1, EEA1) domain,
which binds to membrane lipids, and an UIM (Ubiquitin Interaction Motif) domain that
determines an important role for ESCRT-0 in the initial selection of ubiquitinated cargo at
the endosomal membrane (Hurley & Emr, 2006; Williams & Urb, 2007). Then, ESCRT-0
recruits ESCRT-I, formed by Vps23, Vps28, Vps37 and Mvb12 proteins (Curtiss et al., 2007;
Katzmann et al., 2001). Vps23 also recognizes and binds ubiquitinated proteins through its
terminal UEV (Ubiquitin E2 Variant) domain. ESCRT-I binds to ESCRT-II formed by Vps22,
Vps25 and Vps36 proteins (Babst et al., 2002a). This later protein also displays an ubiquitin-
interacting domain and a recognition region for phosphoinositides binding. Next, ESCRT-II
binds to the ESCRT-III complex composed of Vps2, Vps20, Vps24 and Vps32 proteins (Babst
et al., 2002b). Vps32 associates to Bro1, which recruits Doa4, an ubiquitin hydrolase that
removes ubiquitin from cargo proteins prior to their incorporation into MVB (Kim et al.,
2005; Odorizzi et al., 2003). One of the main functions of ESCRT-III is to concentrate the
MVB cargo in the endosomal inward membrane, and to recruit Vps4, an ATPase that
catalyzes the disassembly of ESCRT complexes from the endosomal membrane to initiate
new rounds of cargo sorting and trafficking, and vesicle formation, and vesicle formation
(Hurley & Emr, 2006; Hurley & Hanson, 2010; Williams & Urb, 2007).
Accessory proteins such as Vta1 and Ist1, regulate Vps4 function (Dimaano et al., 2008;
Shiflett et al., 2004), whereas Vps46 and Vps60 have also been suggested to bind ESCRT-III,
although their precise functions have yet to be determined (Babst et al., 2002b).
1.3 Evolution of the ESCRT machinery
During the evolution from prokaryotic to eukaryotic organisms, some properties were lost
while others were acquired. Among the latter is the ability of eukaryotic cells to incorporate
macromolecules, complexes and other cells through endocytosis (de Souza et al., 2009).
Comparative genomics and phylogenetic studies have determined that the basic features of
intracellular trafficking systems arose very early in eukaryotic evolution (Dacks & Field,

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2007). Similarly, evidence for the existence of MVB-like organelles in diverse primitive
eukaryotes has also been reported (Allen et al., 2007; Tse et al., 2004; Yang et al., 2004).
Lysosomal targeting of ubiquitinated cargo by ESCRT complexes is conserved in animals
and fungi (Leung et al., 2008). Extensive experimental and bioinformatical comparative
analysis of genomic data indicate that ESCRT factors are well conserved across the
eukaryotic lineage (Williams & Urb, 2007). ESCRT-I, -II and III as well as -accessory
proteins are almost completely retained in all studied taxa, indicating an early evolutionary
origin and a near-universal system for cargo trafficking through the MVB pathway.
Particularly, all eukaryotic organisms studied to date have at least an ESCRT-III protein,
suggesting that the minimal ESCRT necessary for MVB formation might be ESCRT-III
(Williams & Urb, 2007). In addition, the number of components of ESCRT-III is greatly
expanded in mammals in comparison to yeast, being Vps46 the most frequent ESCRT-III
multicopy gene product (Dacks et al., 2008).
A common ancestry within the same ESCRT complexes or among them, has been reported
for Vps20, Vps32 and Vps60 proteins (sharing a Snf7 domain), and Vps2, Vps24 and Vps46
proteins (sharing a Vps24 domain). All these proteins are highly similar at sequence level
and are encoded by multicopy genes, probably due to gene amplification events (Leung et
al., 2008). In terms of biological conservation, it seems that several ESCRT components had
to be expanded to provide functional redundancy. Thus, this redundancy would preserve
ESCRT functions in the endocytic MVB pathway even if losses of components were
presented along evolution.
Significantly, the Vps4 ATPase responsible for recycling ESCRT components, is present in
all taxa, indicating a highly conserved mechanism for delivering energy in the system. This
is consistent with recent evidence for an archael origin for Vps4 (Obita et al., 2007).
The most prominent evolutionary variation in the MVB pathway is the restriction of ESCRT-
0 to animals and fungi, suggesting that a distinct mechanism for ubiquitin labeling, signal
recognition and endosomal membrane binding likely operates in the rest of eukaryotic
organisms (Leung et al., 2008).
1.4 Endocytosis and the MVB pathway in parasitic protozoa
Protozoa are a diverse group of single cell eukaryotic organisms, in some of them are
pathogens. Parasitic infections due to protozoa affect millions of people worldwide, causing
a wide range of diseases, high rates of morbidity and mortality each year and an immense
economic burden for public health (Geoff, 1997).
In pathogenic protozoa, endocytosis is a basic mechanism for ingesting host
macromolecules and it has thus been associated to parasite virulence. Previous work based
on ultrastructural, cytochemical, biochemical and molecular studies has shown that
protozoan parasites possess the structural compartments and proteins necessary to perform
endocytosis (de Souza et al., 2009). The extent of endocytic activity varies among different
protozoa and even across various developmental stages. In addition, in trypanosomatids,
the endocytic process is highly active in a well-defined region of the parasite cell surface
called the flagellar pocket (Ghedin et al., 2001). However, only very few studies have been
published to characterize the endocytic MVB pathway in protozoan parasites, some of them
are summarized below.
Giardia lamblia is a protozoan parasite that causes diarrheal infections. It is also one of the
most primitive organisms, with a substantially different endomembrane morphology as


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compared to higher eukaryotes. Although the morphology of membrane-bound vesicles in
Giardia has been previously described, there exists few information about vesicle budding
and fusion (Lanfredi-Rangel et al., 1998). Recently, it was reported that a putative gene
encoding a FYVE domain-containing protein homologous to yeast Vps27 is expressed in G.
lamblia. This protein binds to endosomal membrane phospholipids suggesting the presence
of a MVB pathway in this parasite (Sinha et al., 2010). However, very little is known about
the ESCRT machinery in Giardia (Leung et al., 2008).
Leishmania major, a flagellated parasite provoking leishmaniasis disease, presents a plasma
membrane invagination (flagellar pocket) where the flagellum emerges. This site contains a
complex and highly polarized MVB-like network where endocytosis and exocytosis occur for
crucial exchanges such as nutrient uptake. In this parasite, a Vps4 homologue (LmVps4) has
been characterized using a Vps4 dominant negative mutant in which the highly conserved E
residue required for ATP hydrolysis was substituted by a Q amino acid at position 235. The
LmVps4 mutant protein was accumulated around endocytic vesicular structures and this
provoked a defect in cargo protein transport to the MVB-lysosomes, as it has been reported for
yeast and mammalian Vps4 mutants (Babst et al, 1998; Fujita et al., 2003). Additionally,
LmVps4 is probably involved in Leishmania pathogenicity, since the Vps4 mutant protein also
impaired parasite differentiation and virulence (Besteiro et al., 2006).
Trypanosomes infect a variety of hosts and cause several diseases, including the fatal human
diseases known as sleeping sickness and Chagas disease. In this group of flagellate
protozoa, the trafficking system has been previously characterized (Field et al., 2007).
Trypanosomes contain glycosil-phosphatidylinositol-anchored proteins and
morphologically-related MVB structures, and also exhibit ubiquitin-dependent
internalization of transmembrane proteins for degradation (Allen et al., 2007; Chung et al.,
2004). The functional conservation of the ESCRT system has been confirmed in Trypanosome
brucei. Despite extreme sequence divergence, epitope-tagged Trypanosome TbVps23 and
TbVps28 proteins localize to the endosomal pathway. Knockdown of TbVps23 partially
prevents degradation of ubiquitinated proteins. Therefore, despite the absence of an ESCRT-
0 complex, the MVB pathway seems to function in this parasite, similarly to the yeast and
human systems (Leung et al., 2008).
Members of the Apicomplexan phylum of intracellular parasites, such as Plasmodium
falciparum and Toxoplasma gondii, responsible for malaria and toxoplasmosis, respectively,
contain morphologically unique secretory organelles termed rhoptries that are essential for
host cell invasion, and also display internal membrane-resembling MVB structures
(Coppens & Joiner, 2003; Hoppe et al., 2000). In T. gondii, it has been hypothesized that the
MVB pathway could intersect with the rhoptry biogenesis one. To explore this, wild type
(PfVps4) and mutant (PfVps4E214Q) P. falciparum Vps4 proteins were independently
overexpressed in T. gondii. As expected, PfVps4 was located in T. gondii vesicular structures,
whereas PfVps4E214Q was found in aberrant organelles where rhoptries proteins were also
present, indicating that the secretion pathway could be disrupted by the altered Vps4
protein. These findings suggest that MVB formation may occur in T. gondii and P. falciparum
and that it could be affecting the secretory route too (Yang et al., 2004).
During host cell infection, P. falciparum lives within a special compartment known as the
parasitophorous vacuole. For the parasite to survive and multiply, molecules from the host cell
cytoplasm cross the parasitophorous vacuole membrane and trigger signals for the endocytic
process. Despite the scarce information being available for supporting a feasible relationship
between the MVB pathway and the mechanism of nutrient uptake and intracellular

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phagotrophy (the ability to ingest portions of host cytoplasm) through the parasitophorous
vacuole, it may be possible that these two processes are related (de Souza et al., 2009).
E. histolytica, which causes amoebiasis, destroys almost all human tissues through
macromolecules participating in adherence, contact-dependent cytolysis and proteolytic and
phagocytic activities. A well-characterized protein involved in these key events is
EhADH112 (Garca-Rivera et al., 1999). Interestingly, this protein is located at MVB-like
structures in E. histolytica trophozoites and is structurally related to Bro1 (Bauelos et al.,
2005), an accessory protein that interacts with the ESCRT-III complex in yeast. Recently, our
research group reported the presence of a set of 19 putative ESCRT proteins in this parasite
and characterized a yeast Vps4 homologue by analyzing its ATPase function and
relationship to parasite virulence in wild type and mutant cells (Lpez-Reyes et al., 2010).
Results derived from these studies strongly suggest that E. histolytica possesses a well
conserved ESCRT machinery.
2. Experimental approaches for the identification and characterization of
ESCRT proteins
The ESCRT components involved in mediating endosomal MVB sorting of ubiquitinated
proteins have been identified and characterized by several methodologies.
Initially, over 70 vps genes required for the vacuolar transport of proteins were identified by
genetic screening in yeast (Bonangelino et al., 2002; Bowers et al., 2004). At this moment,
only 20 of these genes are known to be functionally involved in yeast MVB formation.
In addition, the structure and function of putative binding domains present in ESCRT
components have been characterized using recombinant proteins and site-directed
mutagenesis. In particular, ubiquitin recognition and binding to ESCRT complexes by
proteins such as Hse1 and Vps27, Vps23 or Vps36 were elucidated by using crystallographic
structures of recombinant proteins that associate or not, to ubiquitin. The same
methodologies have been used for characterizing lipid binding domains such as the FYVE
motif, present in Vps27, and for positively charged regions with affinity to
phosphoinositides, such as those exhibited by Vps36 and Vps24 (Misra & Hurley, 1999;
Pornillos et al., 2002; Stahelin et al., 2002; Sundquist et al., 2004).
The yeast two-hybrid system is an assay to examine protein interactions. This system
includes the construction of a bait protein containing a DNA binding domain, which
hybridizes to a prey protein with an activation domain. The expression of the reporter gene
means that the proteins of interest interact with each other since the activation domain
promotes the transcription of the reporter gene (Gietz et al., 1997). On the other hand, pull-
down assays are performed either to prove a suspected interaction between two proteins or
to investigate unknown proteins or molecules that may bind to a protein of interest
(Kaltenbach et al., 2007). Alternatively, affinity purification of histidine- or glutathione-
succinyl-transferase-(GST)-tagged bait proteins can be performed via immobilized affinity
chromatography. The bait protein (or ligand) is captured to a solid support (beads) by
covalent attachment to an activated beaded support or through an affinity tag that binds to a
receptor molecule on the support (Pandeya & Thakkar, 2005).
In yeast, Bro1 binding to Vps32 was discovered by two-hybrid experiments, whereas Bro1
association to Vps4 was revealed by GST pull-down experiments. Additionally, using both
methodologies, interactions between Vps20 and Vps28; Vps20 and Vps22; and Vps22 and


296
Vps28, were identified. Moreover, protein-protein interactions for ESCRT assembly have
been evidenced by yeast-two-hybrid assays, affinity purification or both methods (Vps20
with Vps25 and Vps36; Vps27 with Hse1; Vps4 with Vps32; Vps22 with Vps25; and Vps22
with Vps36) (Bowers et al., 2004).
Another strategy to study protein functions is via dominant negative (DN) mutants.
Mutations are changes in a genomic sequence and sometimes their expression is dominant
over the wild-type protein synthesis in the same cell. Usually, DN mutants can still interact
with the normal partner proteins thus blocking the functions of the wild-type protein. To
improve our knowledge on the ESCRT model, several DN mutants for Vps proteins have
been generated, including Hrs, Vps27, Vps23, Vps20 and Vps4 (Kanazawa et al., 2003; Li et
al., 1999; Fujita et al., 2003).
Research using such strategies has increased our knowledge on the identity, structure,
function and biological relationships of several molecules participating in the protein sorting
through the endosomal MVB pathway. However, complementary experimental efforts need
to be performed to better understand this cellular process.
3. Computational research on protein biology
One of the most familiar applications of bioinformatics is the comparison of the amino acid
sequence from a query protein against the amino acid sequence of a protein previously
characterized in structure and function, to theoretically elucidate whether they are related.
This approach gives insights into functional similarities and evolutionary relationships
deduced from the presence of common structural features (Sding, 2005).
Similarity and homology are two important concepts in the bioinformatical analysis of
protein sequences. Similarity is a quantitative measure between two or more related amino
acid sequences. By contrast, homology is a qualitative measure which indicates if two or
more proteins are evolutionarily related or derived from a common ancestor (Claverie &
Notredame, 2006). Protein sequences are usually submitted, annotated and stored in
databases that allow their comparison and analysis by certain software.
In general, a database is a digital system that organizes, stores and easily retrieves large
amounts of data. Currently, several genome and proteome databases are freely available for
studying protein biology. However, the sheer amount of data makes highly difficult to
manually interpret it. Therefore, databases require supplementary and incisive
computational tools in order to understand the information.
One of the most recognized databases is the UniProt Knowledgebase (UniProtKB,
http://www.uniprot.org/). The UniProtKB is the central hub for the collection of functional
information on annotated proteins. The UniProtKB consists of a section containing
manually-annotated records with information extracted from literature and curator-
evaluated computational analysis (UniProtKB/Swiss-Prot), and a section with
computationally analyzed records that await full manual annotation (UniProtKB/TrEMBL).
Manual annotation consists of a critical and continuously updated review of experimentally
proven or computer-predicted data about each protein by an expert team of biologists.
The UnipProtKB captures the mandatory core data for each entry (amino acid sequence,
protein name, description, taxonomic data and citation information) and supplementary
information derived from experimental evidence or computational data.

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More than 99% of the protein sequences provided by UniProtKB comes from coding
sequences translation and related data submitted to the public nucleic acid databases,
including the European Molecular Biology Laboratory (EMBL) Bank, the GenBank (USA)
and the DNA DataBank of Japan (DDBJ). Taking advantage of the information as much as
possible, there are a number of computational tools to finally interpret databases, some of
them are briefly described below.
The Expert Protein Analysis System (ExPaSy) is a proteomics server from the Swiss Institute
of Bioinformatics that analyzes protein sequences and structures and contains genome
databases for several organisms ranging from Archae to human (http://expasy.org/
tools/proteome). It has several tools useful to depict primary, secondary and tertiary
protein structures and to determine putative postranslational modifications, among others.
The Basic Local Alignment Search Tool (BLAST) is an algorithm for comparing primary
biological sequence information, such as amino acid sequences of different proteins or
nucleotides of distinct DNA sequences. A BLAST search enables a researcher to compare a
query sequence with data existing in sequence libraries or databases, and to identify the
sequences that resemble the query sequence above a certain threshold. The main idea of
BLAST is that there are often high-scoring segment pairs (HSP) contained in a statistically
significant alignment. BLAST searches for high scoring sequence alignments between the
query sequence and sequences from genome databases, using a heuristic approach that
approximates the Smith-Waterman algorithm (Altschul et al., 1990). The BLASTP program,
which compares protein queries to protein databases, is a heuristic model that attempts to
optimize a specific similarity measure. The goal of this tool is to find regions of sequence
similarity. These regions can yield clues about the structure and function of the novel
sequence and its evolutionary history and homology by comparison to other sequences in
databases (Henikoff & Henikoff, 2000). To produce a multiple sequence alignment from the
BLASTP output, this program simply collects all database sequence segments that have been
aligned to the query with an expectation value (E-value) below a threshold by a default set
to 0.001. Thus, the lower the E-value, the greater the similarity between the input and the
match sequences will be. An E-value < e-3 of an alignment means that the alignment is
highly unique and not due to error (http://bips.u-strasbg.fr/fr/Tutorials/Comparison/
Blast/blastall.html). As an alternative for accurate searches of query sequences, the Position
Specific Iterative (PSI)-BLAST program iteratively searches for one or more proteins
databases to find sequences similar to one or more protein query sequences.
ClustalW is also a widely used multiple sequence alignment computer program
(http://align.genome.jp/). In many cases, the input set of query sequences is assumed to
have an evolutionary relationship, share a lineage and descend from a common ancestor.
This algorithm is usually supplemented by the BOXSHADE application
(http://www.ch.embnet.org/software/BOX_form.html). BOXSHADE is a program for
creating good looking printouts from multiple-aligned protein or DNA sequences.
BOXSHADE does not produce alignments by itself, it has to take as input a file preprocessed
by a multiple alignment program or a multiple file editor such as ClustalW. In the standard
BOXSHADE output, identical and similar residues in the multiple-alignment chart are
represented by different colors or shadings.
3.1 Computational tools for predicting protein domains
Protein domains, defined as the independent folding units within a polypeptide, are also
understood as the functional and evolutionarily conserved modules of protein families.


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The Pfam protein family database is a large collection of multiple sequence alignments that
is generated by probabilistic models known as hidden Markov models (HMM)
(http://www.sanger.ac.uk/resources/databases/pfam.html). The Pfam database contains
information about protein domains and families. For each family in Pfam, one can look at
multiple alignments, view protein domain architectures, examine species distribution, and
follow links to other databases and view known protein structures.
Despite the increasing volume of biochemical and molecular literature on protein data, Pfam
contains the essential information about major protein domains for the understanding of the
ever more complicated biological landscape.
Since the ClustalW and BOXSHADE programs could be useful to identify conserved
residues and similar regions among amino acid sequences, they also allow the prediction of
putative domains in a protein or group of proteins of interest.
3.2 Computational approaches for predicting secondary protein structure
Secondary structure refers to highly regular local sub-structures within a molecule. The
secondary structure of a protein is defined by patterns of hydrogen bonds between the
main-chain peptide groups, leading to several recognizable protein domains, such as alpha
() helices and beta () sheets (Offer et al., 2002).
So far, several algorithms have been described for predicting secondary protein structures,
one of them being Jpred (Cole et al., 2008). Jpred uses a 3-iteration PSI-BLAST search to
obtain sequences from existing databases for predicting secondary structures. Jpred now
includes Jnet, a neural network method also for secondary structure prediction. The Jnet
algorithm works by applying multiple sequence alignments, alongside PSI-BLAST and
HMM profiles (Cuff & Barton, 1999). The updated Jnet algorithm provides -helix and -
sheet predictions at an accuracy of 81.5% (Cole et al., 2008).
3.3 Computational algorithms for predicting tertiary protein structure
The tertiary structure of a protein refers to the three-dimensional arrangement of a single
protein molecule. The -helices and -sheets are folded into a compact structure due to non-
specific hydrophobic interactions. However, this structure is stable only when the parts of a
protein domain are locked into place by specific tertiary interactions, such as salt bridges,
hydrogen bonds, and the tight packing of side chains and disulfide bonds (Peng & Kim,
1994).
The Protein Data Bank (PDB) contains information about experimentally-determined
structures of proteins and nucleic acids, and complex assemblies
(http://www.pdb.org/pdb/home/home.do). The Resource for Studying Biological
Macromolecules curates and annotates the PDB data according to agreed upon standards
and also provides a variety of tools and resources. Interestingly, the PDB is a repository for
three dimensional structural data of proteins (typically obtained by X-ray crystallography or
Nuclear Magnetic Resonance spectroscopy) submitted by biologists and biochemists from
around the world.
The PDB is a key resource in areas of structural biology, such as structural genomics.
Contents of the PDB are thought to be primary data, and currently there are hundreds of
derived databases that categorize data differently.
The Phyre (Protein homology/analogy recognition engine) webserver is a powerful
computational tool that uses profile-profile matching algorithms to considerably improve

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protein predictions (Kelley & Stenberg, 2009). The Phyre platform follows the most
successful general approaches for predicting the structure of proteins, which involve the
detection of homologues of a known three dimensional structure, the so-called template-
based homology modeling and fold-recognition. Practical applications from three
dimensional protein structure predictions include guidance on functional hypothesis, the
selection of mutagenesis sites and the design of rational drugs, among others.
The Phyre server uses a library of known protein structures taken from the SCOP (Structural
classification of proteins) database and augmented with newer depositions in the PDB.
Sequences of each of these structures are scanned against a non-redundant sequence
database and a profile is constructed and deposited in a fold library. The known and
predicted secondary structures of these proteins are also stored in the fold library. A user-
submitted sequence follows the same process. Five iterations of PSI-BLAST are used to
gather both close and remote sequence homologues. The pairwise alignments generated by
PSI-BLAST are combined into a single alignment with the query sequence as the master.
Following the profile construction, the secondary structure of the query is predicted using
three distinct programs (Psi-Pred, SSPro and Jnet). Subsequently, both profile and secondary
structure, are scanned against the fold library using a profile-profile algorithm that returns a
score. Scores are fitted to an extreme value distribution to generate an E-value. The top ten
highest scores are then used to construct full three-dimensional models for the query. Where
possible, missing or inserted regions caused by deletions or insertions in the alignment are
repaired using a loop library and reconstruction procedures.
An alternative program widely used to model tertiary protein structures is SWISS-MODEL.
SWISS-MODEL is a fully automated protein structure homology-modeling server accessible
via the ExPASy web server or from the DeepView program
(http://swissmodel.expasy.org/). The purpose of this server is to make protein modeling
accessible to all biochemists and molecular biologists worldwide by providing tools for
protein structure accurate predictions.
Once a tertiary structure has been modeled, it is sometimes necessary to get access into a
model viewer. Jmol is a free open-source viewer for chemical three dimensional structures
that is written in Java (so it runs on Windows, Mac OS X, Linux and UNIX systems). Jmol
returns a representation of a molecule that may be used as a teaching tool, or for research
e.g. in chemistry and biochemistry. The most notable feature is an applet that can be
integrated into web pages to display molecules in a variety of models: ball and stick,
space filling, ribbon, etc. (http://jmol.sourceforge.net/download/).
4. ESCRT protein survey in protozoan parasites with bioinformatical tools
By using a bioinformatical screening and comparative genomic analysis, we confirmed in
this work the presence of ESCRT representatives in unrelated groups of unicellular parasites
of medical importance belonging to the following taxa: Entamoebidae (Entamoeba),
Diplomonadida (Giardia), Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium),
and Kinetoplastida (Trypanosoma and Leishmania).
First, we obtained yeast or mammalian amino acid sequences for ESCRT-0 to -III and -
associated proteins from the UniProtKB database. Then, the retrieved sequences were
used as probes to screen the Eukaryotic Pathogen database (EuPathDB version 2.9,
http://eupathdb.org/eupathdb/). The EuPathDB has been developed as a Bioinformatics
Resource Center and constitutes an integrated genome database covering eukaryotic


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pathogens of the genera Cryptosporidium, Giardia, Entamoeba, Leishmania, Plasmodium,
Toxoplasma, Trichomonas and Trypanosoma, among others. This portal offers an entry point
to all these resources, and the opportunity to leverage orthology (structural
correspondence or similarity of genes or proteins in different species due to a common
ancestor origin) for searches across genera in an interface that is functional, user-friendly
and sophisticated.
Using yeast ESCRT protein sequences as queries in the EuPathDB resource for each parasite
genome, the BLASTP program reported several amino acid sequences for each pathogen.
When no matches were found, human corresponding ESCRT protein sequences were used
as queries. Putative parasite ESCRT homologous sequences were selected with the following
criteria: i) at least 20% identity and 35% similarity to the query sequence, ii) E-value lower
than 0.002, and iii) absence of stop codons in the coding sequence. Furthermore, all
recovered sequences were subjected to reverse BLAST analysis in the ExPaSy server to
identify related proteins from genome databases. A candidate was taken into consideration
if reverse BLAST recovered the original query within the top five hits. Failure to complete
these tests resulted in a not determined assignment.
BLAST results showed that all parasites studied here contain putative protein sequences
representing the ESCRT-0 to -III and -accessory proteins involved in the endocytic MVB
pathway. In Table 1, we summarized the results derived from our parasite ESCRT genomic
survey in comparison to ESCRT members previously reported in yeast or human. The major
noticeable feature was the high conservation of ESCRT components in all taxa, as previously
reported (Leung et al., 2008). As noticed, Entamoeba histolytica and Leishmania major contain
the most represented and conserved ESCRT machinery among parasites, with 19 ESCRT
components. Meanwhile, Trypanosoma cruzi and Plasmodium falciparum displayed 15 and 14
ESCRT putative proteins, respectively. By contrast, we only found 9 out of the 20 ESCRT
proteins in Toxoplasma gondii and Giardia lamblia.
Ubiquitin-label recognition is the signal for cargo protein entrance towards degradation
through the endosomal pathway (Bowers et al., 2004). Rsp5 and Bul1 proteins mediate
ubiquitin-attachment to cargo proteins in yeast. Here, bioinformatical approaches revealed
that ubiquitination seems to be mediated by Rsp5 rather than Bul1 homologues, since Rsp5-
like proteins were present in all protozoan genomes.
Unlike preceding work, we found at least one ESCRT-0 representative for each parasite,
indicating that proteins recognizing ubiquitin signals could be participating in cargo sorting
in these protozoa. ESCRT-I and -II were the least represented complexes among all
parasites, suggesting that some taxa members could have lost specific components along
ESCRT evolution. However, we cannot exclude that the lack of individual ESCRT
components might be the result of malfunctionings in gene or protein detection, more than a
real absence of the protein. In particular, failures have been frequently reported for Giardia
due to difficulties to recover candidate orthologues in its extremely divergent genome.
To the best of our knowledge, there is no sequenced eukaryotic genome without an ESCRT-
III-related gene. Moreover, the size of the subset of ESCRT-III-related genes is greatly
expanded in higher eukaryotes such as mammals, compared to yeast. As a consequence, it
has been hypothesized that the ESCRT-III complex might be the minimal ESCRT unit for
MVB formation (Williams & Urb, 2007). Consistently, our results revealed at least two
ESCRT-III representatives in each parasite genome analyzed.
Regarding the ESCRT-accesory proteins, the most conserved sequences among all parasites
were the Rsp5, Vps4, Vps46, Doa4, Vta1 and Bro1 homologues, in contrast to Ist1, which was
only present in trypanosomatids.

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Taken together, our in silico results support the existence of a seemingly conserved ESCRT
machinery for endosomal protein trafficking through the MVB pathway in protozoan
parasites.

Table 1. Comparison of ESCRT machineries from parasitic protozoa.
The presence (+) of homologous proteins is based on data obtained by BLAST searches from
protein sequence databases at NCBI, UniProtK and EuPathDB, as described in the text.
Proteins apparently absent (-) from complete genome sequencing projects are indicated.
4.1 Characterization of the ESCRT machinery in E. histolytica
Our previous work, using comparative genomics for predicting ESCRT proteins in E.
histolytica, provided valuable insights into the existence of a highly conserved ESCRT
machinery in this parasite. Lpez-Reyes et al. (2010) reported a set of 19 putative ESCRT
proteins representing from ESCRT-0 to -III and -associated proteins (Table 2). Moreover,
earlier characterization of ubiquitin genes and -transcripts and demonstration of an
ubiquitin-conjugating system, together with our finding of a putative Rsp5 ubiquitin ligase
(EhRsp5) E. histolytica provided additional support for the presence of at least one candidate
that possibly mediates ubiquitin attachment to cargo molecules prior to their internalization
into endosomes (Wstmann et al., 1996).
Previous work has provided knowledge into the architecture, membrane recruitment and
functional interactions of the ESCRT machinery through multiple domains that have been
shaped along evolution. These scaffolds serve as gripping tools for recognizing cargo
proteins, membrane lipids, ESCRT components and accessory proteins along the MVB route
(Hurley & Emr, 2006).


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To dissect the presence of putative ubiquitin and phosphoinositide binding domains in E.
histolytica ESCRT-like components, we selected ESCRT-0 to -III representatives (EhVps27,
EhVps23, EhVps36 and EhVps24, respectively) presumably containing these structural
features according to their yeast and human homologues and performed multiple sequence
alignments with the ClustalW program.

Table 2. Comparison of E. histolytica, H. sapiens and S. cerevisiae ESCRT machineries.
Data of conserved ESCRT proteins from yeast and human were obtained at NCBI and
UniProtKB databases. Putative E. histolytica ESCRT proteins were retrieved by BLAST searches
at EupathDB and corresponding UniProtKB accession numbers were obtained. Putative
ESCRT proteins of E. histolytica exhibited significant E-values (1.1e-114

to 0.00032) and high
similarity (20 to 62%) to yeast and human ESCRT orthologues. nd, not determined; ----, non-
significant similarity or identity and E-values; S, similarity; I, identity (Modified from Lpez-
Reyes et al., 2010).
Our computational comparative analysis showed that the ESCRT-0 complex, lacks the
characteristic VHS (Vps27, Hrs and STAM) domain of yeast Vps27, required for the protein
interaction with ubiquitin (Williams & Urb, 2007). However, EhVps27 displayed a
(R/K)(R/K)HHCR motif usually found within conserved FYVE domains and necessary for
phosphatidylinositol 3-phosphate (PtdIns3P) binding (Misra & Hurley, 1999). This finding
was also supported by the Pfam database, which reported the presence of a putative FYVE
domain in the EhVps27 amino acid sequence.

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Membrane phospholipids such as PtdIns3P, have been previously implicated in the
regulation of endocytosis and phagocytosis and 12 FYVE-domain containing proteins have
been identified in E. histolytica (Nakada-Tsukui et al., 2009).
The UEV domain present in yeast Vps23 and its human homologue Tsg101 is necessary to
recognize ubiquitin signals in proteins to be sorted into MVB (Pornillos et al., 2002;
Sundquist et al., 2004). Despite a less conserved similarity among analyzed sequences, our
bioinformatics approach suggested the presence of a putative UEV domain at the N-
terminus of EhVps23, also supported by Pfam domain predictions.
According to our current investigation, EhVps36 lacks the yeast NZF and human GLUE
domains previously reported in Vps36 homologues. Both domains have been implicated in
ubiquitin and PtdIns3P binding, respectively. Instead, EhVps36 conserves a N-terminal
positively charged amino acid region. Similarly, the EhVps24 protein exhibits a positively
charged amino acid tract present in almost its full sequence. Since specific binding to
phosphoinositides requires electrostatic interactions between negatively charged
phosphates on lipids and positively charged amino acids in proteins, it is feasible that
EhVps36 and EhVps24 associate to phosphoinositides present at endosomal membranes
(Whitley et al., 2003).
Secondary structure assignments for putative ESCRT proteins of E. histolytica were achieved
by using the Jpred program. In agreement with our previous findings, EhVps27, EhVps36
and EhVps24, and EhVps23 proteins resulted in similar arrangements to yeast Vps27, Vps36
and Vps24 proteins, and human Tsg101, respectively. Furthermore, according to both Phyre
and SWISS-MODEL tertiary structure predictions, the three-dimensional structures of
EhVps27 and EhVps36 matched to yeast Vps27 (PDB code: 1vfy) and Vps36 (PDB code:
1u5t) crystalline structures, respectively. In addition, the Phyre software predicted a
conformational arrangement similar to human Tsg101 (PDB code: 1s1q) and CHMP3 (PDB
code: 2gd5) proteins for EhVps23 and EhVps24, respectively.
Altogether, our results indicate the presence of putative structural and conformational
features for ubiquitin and lpid binding in representative proteins from the E. histolytica
ESCRT-0, -I, -II and III complexes.
To determine the identity of putative ESCRT-accesory proteins, we first focused on
EhADH112, a protein widely studied by our group and involved in E. histolytica adherence
to and phagocytosis of host cells (Garca-Rivera et al., 1999). In silico analysis of the primary
sequence of EhADH112 together with Pfam protein domain predictions, revealed that
EhADH112 is structurally related to yeast Bro1 and its human homologue Alix. EhADH112
has a conserved Bro1 domain at its N-terminus. In Bro1 and Alix proteins, the Bro1 domain
constitutes the interacting site for Vps32 or CHMP4B, respectively, both components of the
ESCRT-III complex. Experimental approaches demonstrated that E. histolytica parasites
overexpressing only a part of the EhADH112 Bro1 domain, reduced dramatically their
ability to ingest cells, thus providing additional evidence for EhADH112 participation in
phagocytosis (our unpublished results). Furthermore, immunolocalization of EhADH112
and truncated EhADH112 proteins in parasites, using both transmission electron and laser
confocal microscopy, revealed that besides its detection at the plasma membrane and
cytoplasmic vacuoles, EhADH112 is also in MVB-like organelles, whereas the EhADH112
mutant version accumulates in cytoplasmic vesicles. These findings led us to assign a
putative role for the EhADH112 Bro1 domain to recruit proteins to the endosomal
membranes forming MVB. Possibly, Vps proteins from the ESCRT-III complex or some
other molecules could be involved in this event, thus affecting the E. histolytica phagocytosis
process. In order to identify putative interacting partners for EhADH112, we used a


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computational survey for yeast Vps32 or human CHMP4B homologous sequences in the E.
histolytica genome. We found a putative EhVps32 protein whose existence in E. histolytica
was confirmed by further experimental data (Bauelos et al., 2007). According to multiple
sequence analysis and Pfam database predictions, EhVps32 contains a Snf7 domain, present
in all members of the Snf7 family. Additionally, the predicted EhVps32 secondary structure
using the Jpred program, suggested that EhVps32 conserves the characteristic five -helices
present in the Snf7 family protein (Fig. 2A). Using the Phyre program, the tertiary structure
for EhVps32 was modeled. Results showed that the predicted structure of EhVps32 is
related to human CHMP3, a Snf7 family member (Fig. 2B). Since the crystal structure for
CHMP4B has not yet been solved, the program uses by default the CHMP3 crystal structure
as template due to the presence of the highly conserved Snf7 domain. Thus, tertiary
structures for CHMP4B and Vps32 were also modeled using CHMP3 as template (Fig. 2B).
Retrieved results showed that EhVps32 adopts a conformational structure and folding more
similar to CHMP4B than to yeast Vps32 and this is in agreement with the highest similarity
reported for EhVps32 to the human sequence of CHMP4B by BLAST analysis (Table 2).
To confirm the predicted interaction between EhADH112 and EhVps32 proteins, pull down
experiments were perfomed. Assays demonstrated that EhADH112 binds through its N-
terminus to a recombinant protein of EhVps32 fused to GST (our unpublished data).
Since yeast Vps4 and its orthologues have been previously described as key molecules for
ESCRT dissociation and recycling, Lpez-Reyes et al., (2010) characterized the EhVps4 protein
in more detail. Protein domain predictions, as well as tertiary structure modeling and
phylogenetic trees assayed for EhVps4 suggest, that it conserves a typical Vps4 architecture
(Babst et al., 1998) and is more related to protozoan Vps4 homologues than to that of higher
eukaryotes. Biochemical experiments using an EhVps4 recombinant protein and ATP as
substrate, evidenced the ATPase activity of EhVps4 in vitro. As expected, when using a mutant
version of EhVps4, in which an E residue was substituted by a Q amino acid, the ATPase
activity was reduced. Furthermore, E. histolytica parasites overexpressing the EhVps4 mutant
protein displayed reduced virulence properties, suggesting a role for EhVps4 in parasite
pathogenicity, probably related to its participation in the endocytic pathway.
5. Challenges and perspectives
Our previous results obtained via bioinformatical tools and biochemical experiments, allow
us to propose a model for the ESCRT machinery in E. histolytica (Fig. 3). Since we found the
EhVps27 component of the ESCRT-0 complex, we suggest that it may initiate the MVB
sorting process. Additionally, EhVps27 has a FYVE domain that possibly mediates protein
binding to the endosomal membrane. However, EhVps27 lacks the UIM domain, important
for the initial selection of ubiquitinated cargo, probably by EhRsp5. Perhaps, EhVps23,
through its UEV motif, or another unidentified protein could be recruiting cargo proteins to
endosomes. Furthermore, the EhVps23 UEV domain could associate toEhVps27 and other
components of the ESCRT-I complex, which includes the EhVps28 and EhVps37 proteins.
Then, ESCRT-I binds to ESCRT-II (formed by EhVps22, EhVps25 and EhVps36 proteins).
Although EhVps36 does not exhibit an ubiquitin-interacting domain as yeast homologues,
this protein contains a recognition region for phosphoinositides that presumably would
allow ESCRT-II attachment to the endosomal membrane. Next, ESCRT-II binds to the
ESCRT-III complex, which contains the overall components previously described for yeast.
Interestingly, similarly to yeast Vps20, EhVps20 has a myristoylated modification that
facilitates ESCRT-III insertion into the endosomal membrane. Then, ESCRT-III interaction

305

Fig. 2. Structural comparison of Vps32 homologues.
(A) At the top, a schematic representation for human CHMP4B, yeast Vps32 and E.
histolytica EhVps32 proteins is shown. Numbers indicate amino acids for each protein. All
proteins contain conserved Snf7 domains, present in the Snf7 family proteins. Vps32
orthologues belong to the ESCRT-III complex and have been described as the interacting
partners of Bro1 domain-containing proteins. At the bottom, a multiple sequence alignment
for Vps32 homologues is shown. Hs, H. sapiens; Sc, S. cerevisiae; and Eh, E. histolytica. Black
boxes, identical amino acids; grey boxes, conserved substitutions; and open boxes, different
residues. Numbers at left are relative to the position of the start codon in each protein. The
Jpred secondary structure prediction program revealed that EhVps32 folds into five -
helices (green horizontal cylinders) as it has been reported for Vps32 homologues. (B)
Tertiary protein structure for H. sapiens, S. cerevisiae and E. histolytica Vps32 homologues.
Modeling was done using the Phyre program with the crystal structure of human CHMP3
as template.


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Fig. 3. Model for the role of the ESCRT machinery in E. histolytica within the endosomal
MVB pathway.
In E. histolytica, the EhRsp5 protein could be responsible for cargo protein ubiquitination.
Then, the EhVps27 protein could initiate the MVB process. Similar to yeast Vps27, EhVps27
has a FYVE domain that binds PtdIns3P allowing endosomal membrane attachment.
However, EhVps27 lacks the UIM domain, important for ubiquitin recognition in cargo
proteins. Instead, EhVps23 could be mediating this event through its UEV motif.
Subsequently, EhVps27 binds to the ESCRT-I complex through EhVps23. Then, EhVps36 by
its positively charged region binds to PtdIns3P, facilitating the ESCRT-II attachment to
endosomal membranes. E. histolytica contains all ESCRT-III components which belong to the
Snf7 family of proteins. In addition, it has several accessory proteins, including the
EhADH112 (a Bro1 domain-containing protein), EhDoa4 (deaubiquitinating enzyme that
removes ubiquitin from cargo) and EhVps4 (an ATPase) proteins. Finally, as in yeast,
EhVps4 may play a critical role in catalyzing the dissociation of ESCRT from the endosomal
membrane in order to start new rounds of cargo protein sorting through MVB.
with accessory proteins could be mediated by EhVps32. In fact, EhVps32 could associate to
EhADH112 through its putative N-terminal Bro1 domain (our unpublished data). Besides,
EhADH112 could also be recruiting another accessory molecule, the EhDoa4 ubiquitin
hydrolase, removing ubiquitin from cargo prior MVB internalization. Finally, the EhVps4
ATPase might catalyze the disassembly of the ESCRT complex from the endosomal

307
membrane to initiate new rounds of cargo sorting and vesicle formation. Possibly, EhVta1
may have a role in regulating EhVps4 function.
Of note, E. histolytica possesses a conserved ESCRT machinery. However, the study related
to ESCRT functions and putative interactions along the MVB pathway needs to be
corroborated by experimental approaches.
6. Conclusions
Bioinformatics, the application of statistics and computer sciences to molecular biology,
entails the creation and advancement of databases, algorithms, computational and statistical
techniques and theory to solve formal and practical problems arising from the management
and analysis of biological data. In this chapter, we used bioinformatics to analyze the ESCRT
protein machinery possibly participating in parasitic protozoa endosomal pathways, with
particular attention on the E. histolytica case.
The ESCRT machinery comprises a set of protein complexes that regulate recognition,
sorting and trafficking of monoubiquitinated proteins into MVB compartments towards
lysosome degradation. Previous work has shed light on molecular details underlying the
assembly and regulation of ESCRT in yeast and human. Here, we took advantage from
eukaryotic pathogen genome database availability and bioinformatics tools to identify
proteins representing putative ESCRT components in protozoan parasites of medical
importance. We found representative proteins for ESCRT-0, -I, -II, -III and -accesory proteins
in almost all protozoa examined, being E. histolytica and L. major the parasites in which
ESCRT components were the most represented. Despite these findings, several issues need
to be experimentally addressed to finely determine the structure and function of ESCRT
proteins and their putative role during endocytosis in these parasites.
In E. histolytica, we found a highly conserved ESCRT machinery with 19 putative
components representing all complexes. These findings have been experimentally confirmed
by determining the expression of most ESCRT gene transcripts (Lpez-Reyes, et al., 2010).
Furthermore, our current in silico results suggest that some E. histolytica ESCRT-0 to -III
components contain putative FYVE or ubiquitin binding domains, both important to recruit
cargo molecules to endosomal membranes. In addition, our computational analysis together
to previous functional characterization of putative E. histolytica ESCRT-accessory proteins,
strongly suggest the presence of a Bro1-domain containing protein (EhADH112), its putative
interacting partnership, EhVps32, and an ATPase (EhVps4) that may be responsible for
energy-dependent ESCRT disassembly. Of note, tertiary structure modeling of EhVps32
supported our experimental findings on EhADH112 binding to EhVps32, proving the value
of bioinformatical approaches. Therefore, our overall results provide significant evidence for
a conserved role of the E. histolytica ESCRT machinery in the MVB endocytic pathway.
In summary, bioinformatics and experimental approaches can improve our understanding
on evolutionary implications of the MVB sorting pathway in E. histolytica, L. major, T. cruzi,
P. falciparum, T. gondii and G. lamblia and also for elucidating its possible relationship to
parasite pathogenicity and virulence.
Although some limitations exist due to incompleteness of experimental data, we conclude
that computational methods have a reasonable prediction accuracy and provide invaluable
basis for further experimental validation.


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7. Acknowledgements
Authors would like to thank Dra. Rossana Arroyo, Dr. Jaime Ortega and Dr. Michael
Schnoor for providing their comments on the manuscript and Alfredo Padilla-Barberi for
efforts in the artwork.
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15
Structural Bioinformatics Analysis of
Acid Alpha-Glucosidase Mutants with
Pharmacological Chaperones
Sheau Ling Ho
Chinese Culture University
Taiwan
1. Introduction
Most lysosomal storage disorders (LSDs) are usually inherited, caused by the deficiency of a
single lysosomal hydrolase, leading to the accumulation of the corresponding substrate.
LSDs can also result from mutations in proteins involved in the intracellular trafficking of
lysosomal enzymes (Carrell & Lomas, 1997, Kopito & Ron, 2000, Selkoe, 2003, and Arakawa
et al., 2006). Indeed, LSDs are considered as a group of more than sixty diverse inherited
disorders. Each of the diseases is due to a specific enzymatic defect (Hodges & Cheng, 2006,
Raben et al., 2009). Pompe disease is one of these LSDs through point mutations (single wild
type amino acid substitutions) in the gene that encodes for acid -glucosidase (GAA). The
resulting total or partial deficiency of lysosomal acid -glucosidase triggers glycogen to
accumulate in lysosomes (Alberts et al., 2002, Raben et al., 2002, Bernier et al., 2004, Kroos et
al., 2008).
Recently, various small molecule pharmacological chaperones have been discovered to
increase stability of such mutant proteins and facilitate their efficient trafficking of
lysosomal enzymes. As such, it pointed the way to a new therapeutic approach in LSDs
treatment. In this study, we are concerned with revealing the mechanism and accurate
structures underlying the defects in the folding behaviors of the involved enzymatic protein
mutants, also the way in which they interact with small molecule pharmacological
chaperones.
The pharmacological chaperone 1-deoxynojirimycin (DNJ) showed improvement in the
treatment of Pompe disease. Yet, experimental data had shown that only a number of GAA
mutants responded well to this pharmacological chaperone (Hirschhorn & Reuser 2001,
Petsko & Ringe, 2004, and Chaudhuri & Paul 2006, Sugawara et al., 2009, Flanagan et al.,
2009). In an effort to improve the stability of mutant enzymes, the understanding on the
molecular interaction between the enzyme and the chaperones is very important. Since
neighboring residues share physical characteristics, we undertook a detailed study of the
surroundings of GAA variants in the structures (Zvelebil, et al., 1987). Thus, we herein aim
at discriminating between structural, as opposed to, GAA mutants, based on analysis of
their local environments.
Despite the absence of crystallographic data of human acid alpha-glucosidase, we reviewed
recently published papers to construct a structural model of human maltase-glucoamylase


314
(MGAM) through homology modeling using the structural information (PDB ID: 2QLY) as a
template. Note that there are approximate 44% amino acid sequence identities between the
GAA and template. Based on the sequence alignment and the structural mode, our structural
model, GAA residues (84-952) were threaded on to the MGAM template. The active site region
for both GAA and MGAM overlaid well and the key catalytic residues had high similar spatial
alignment (D518/D616 and D445/D542 in GAA and template respectively).
This study involved active site analysis that we applied the proposed model to reveal
whether any conformational changes take place at the active site of GAA mutants and
molecular docking studies on DNJ which we presented the geometry of the binding site of
the complexes of GAA/DNJ and GAA mutants/DNJ. These were done by visual inspection
of the atomic models looking at the interaction between the human GAA variants and
chaperones, in terms of both binding energy and spatial orientation of the active site.
Structural studies should be useful in improving our understanding of enzyme protein
stability, molecular recognition and binding and then will help us to further elucidate the
molecular basis of Pompe diseas.
2. Methodology
2.1 Structural modelling of the wild-type and mutant human acid -glucosidase
A structural model of wild-type human acid -glucosidase was built using molecular
modeling software, MIFit (a cross-platform interactive graphics application for molecular
modelling), and Molecular Operating Environment, MOE (CCG-Chemical Computing
Group Inc.), by means of homology modeling. The structural of human intestinal maltase-
glucoamylase (PDB: 2QLY) was used as a template and then energy minimization was
carried out. The root-mean-square gradient (RMSD) was computed in terms of all the atoms
in a protein backbone and the value was less than 0.6 which is indicative of considerable
structural similarity.
More than hundred different GAA mutations know to cause Pompe disease are predicted to
produce full-length proteins corresponding to a single amino acid substitution. Thus, based
on the wild-type human acid -glucosidase model, the structural models of mutants
incorporating the amino acid substitutions were constructed using MIFiT. And the initial
model was further refined by energy minimization. However, because of the low amino
acids sequence identity between the human acid -glucosidase and template, the
investigations were restricted to a limited region of the enzyme protein.
2.2 Molecular docking
2.2.1 Preparation of ligand
The initial structure of the pharmacological chaperone 1-deoxynojirimycin (DNJ) (Figure 1)
for the docking was generated using ChemDraw Ultra Version 9.0 (CambridgeSoft Corp.).
And then geometry optimized ligands were prepared using MOE.
2.2.2 Docking
According to the effect of DNJ on responsive GAA mutants, six severe effects of GAA
variants (G377R, A445P, L552P, Y575S, E579K, and H612Q) and wild-type GAA were chosen
as the receptor for docking (Flanagan et al., 2009). Enzyme proteins and ligand structures
were imported into MOE 2010.10 where three-dimensional structures were generated using
a course energy minimization protocol and the MMFF94x force field (Halgren, 1996, 1999).
Acid Alpha-Glucosidase Mutants with Pharmacological Chaperones

315

Fig. 1. 3D and 2D visualization of DNJ.
2.2.3 Structural analysis of GAA mutation
To exam the effect of atoms, each mutant model was superimposed on the wild-type
structure on the basis of the C atom by the least-square-mean fitting method (Matsuzawa
et al., 2005 and Saito et al., 2008). We assumed that the structure was influenced by an amino
acid substitution when the position of an atom in a mutant differed from that in the wild-
type structure, thus, such substations were expected to affect neighboring residue and to
locally affect the electrostatic surface of the enzyme.
3. Results and discussion
3.1 Structure modelling of human GAA
3.1.1 Wild-type
As the results showed, our constructed wild-type model of GAA appears to be composed of
five domains: a trefoil type- domain (residues 89135), an N-terminal b-sandwich domain
(residues 136346), a catalytic (/)
8
barrel domain (residues 347723) with two inserted
loops, which include insert 1 (residues 444491) and insert 2 (residues 522567) protruding
out between 3 and 3, and between 4 and 4, respectively, a proximal C-terminal domain
(residues 724818) and a distal C-terminal domain (residues 819952) (Figure 2). The key
catalytic activity (D518 and D616) (Hermans et al., 1991, Sugawara et al., 2008 and Sugawara
et al., 2009) and sequence motifs of family 31 glycosyl hydrolases were well conserved
(Davies & Henrissat, 1995, and Lovering et al., 2005).
The proposed active-site pocket here was composed to residues of residues W376, W402,
D404, I441, D443, W481, W516, D518, M519, F525, R600, W613, D616, D645, F649, and H674
(see Figure 3). Like many other sugar-binding enzymes, there were a lot of hydrophobic
residues lining the active-site pocket, including W376, W402, I441, W481, W516, F525, W613,
and F649.
3.1.2 Wild-type vs. mutants
The six mutant forms of GAA which responded to DNJ severely were superposed with
wild-type. After the structure was superimposed, RMSD was computed in terms of the
active-site pocket between the wild-type and mutants and the value were found to be less
than 0.8 respectively in between. These varied situations were illustrated in Figure 3.


316

Fig. 2. GAA structural model. A ribbon diagram of GAA structural model. The orange
shallow circle area represents the active-site pocket.

Fig. 3. A close-up view of the active-site pocket (W376, W402, D404, I441, D443, W481,
W516, D518, M519, F525, R600, W613, D616, D645, F649, and H674).

317

Fig. 4. Superimposed with the corresponding active-site pocket of the wild-type and six
mutant variants GAA. The conserved catalytic residues D518/D616 are circled in red. Of
GAA variant (G377R), Try turned forwards in the active site.
The comparison results were shown that no significant changes in the conformations of
amino acid residues that comprise the active site and mutations of the key catalytic residues
were conserved but when mutated as G377R, Try veered forward in active site. This might
imply that new drugs can be designed or existing drugs can be modified based on its
interaction with the new tyrosine residue (see Figure 4). This observation rules out the
possibility of a conformational difference between the mutant and the wild-type enzyme as
the derivation cause for the reduction of catalytic activity.
3.2 Docking
Molecular docking is utilized for the prediction of protein-ligand complexes which creates
possible protein-ligand complex geometries. To understand the interaction between the
enzyme and the pharmacological chaperone DNJ, we examined the binding affinity of the
DNJ to the enzyme based on the complex geometry and binding energy. In the complex of
the DNJ and enzyme model (either wild-type or mutants), the DNJ molecule fitted into the
active-site pocket well.
Of the wild-type, residues D404, D518 and D616 were predicted to bind to the hydroxyl
groups and the nitrogen of DNJ through hydrogen bonding inside the active-site pocket.
Residues W376, I441, W481, W516, M519, W613, and F649 might be involved in the
hydrophobic interaction of the DNJ. It is assumed that these residues contribute to the
substrate binding specificity. The active-site pocket was apt for DNJ as to both space and
binding. We also observed that the interactions between DNJ and the active-site pocket
residues of wild-type and mutants; the nitrogen of DNJ was interacted with D518 through
hydrogen bonding. (Figure 5 and Figure 6)
The DNJ fit into the active-site pocket well and a limit space between the nitrogen atom of
DNJ and the wall of the active-site pocket of wild-type GAA and mutant variants
respectively were observed (Figure 7, Figure 8 and Figure 9).


318

Fig. 5. The interaction diagram between DNJ and the wild-type GAA inside the active-site
pocket

Fig. 6. Structure of wild-type GAA bound to DNJ.

319

Fig. 7. Surface representation of the active-site pocket of wild-type GAA with bound DNJ.


320

(a) G377R (b) A445P

(c) L552P (d) Y575S

(e) E579K
(f) H612Q

Fig. 8. Structure of GAA mutant variants bound to DNJ.

321

(a) G377R (b) A445P

(c) L552P (d) Y575S

(e) E579K
(f) H612Q

Fig. 9. Surface representation of the active-site pocket of GAA variants with bound DNJ.
G377R variant shows a larger narrow funnel-shaped region of the active-site cavity.


322
We noticed that a narrow funnel-shaped region of the active-site cavity of wild-type GAA
was smaller compared with that of other mutant variants. Especially, not only G377R
variant showed a larger narrow funnel-shaped region of the active-site cavity compared
with that of wild-type GAA or other mutant variants but also of GAA variant (G377R), Try
turned forwards in the active site. Thus, it should be possible to modify this molecule to
develop a novel derivative suitable for Pompe disease.
The theme of molecular docking is a vital aspect in drug discovery and development.
Molecular docking is utilized for the prediction of proteinligand complexes which predicts
the binding affinity of the ligand to the protein based on the complex geometry. The binding
energies also reflect the binding affinity of a ligand. The docking results were described in
Table 1. The values showed that the binding energy of mutated complex (GAA variants)
was higher than that of wild-type complex. Thus, it is interesting to speculate that increase
in binding energy due to mutation might decrease the binding affinity of GAA towards
DNJ, stabilizing GAA, and modulating its activity.

Ligand
(DNJ)
wild-type
Mutants (GAA variants)
G377R A445P L552P Y575S E579K H612Q
Binding Energy
(kcal/mol)
-149.782 -107.416 -99.904 -130.414 -102.599 -109.852 -95.383
Table 1. Energy values obtained in docking calculation.
Still, these binding energies might not yet sufficient for determining binding affinity of
ligands or drug candidates associations, some other physical effects such as electrostatics,
van der waals, hydrogen bonding, and hydrophobic could affect the binding affinity; those
are also needed to be evaluated.
4. Conclusions
This work involved active site analysis, molecular docking and binding energy studies. We
revealed the mechanism in the folding behaviors of the involved enzymatic protein mutants,
and the way they interacted with small molecule pharmacological chaperones based on
spatial schematics which provided a basis for experimental validation. The validity of this
approach was supported by the identification of some known GAA mutants. Therefore, the
conformational changes detected in the distribution of various residues and their
constituents around various GAA mutants should be useful in improving our
understanding of enzyme protein stability, molecular recognition and binding. Effectively
we have demonstrated the corresponding structural conformations associated with GAA
wild-type and mutants in their three-dimensional environment. The difference in binding
energies might rise due to mutations which could affect the binding affinity of DNJ. And
then, it turns out that the complex structures and energy results presented here may provide
useful consideration in the therapeutic approaches to these diseases as well as in the design
of novel inhibitors associated with sucrose degradation.
5. Acknowledgments
The author is grateful to Dr. Wei-Chieh Cheng for his expert advice and useful discussion
and Dr. Cheng-Yuan Huang acknowledged for exchanging information.

323
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Bioinformatics Domain Structure Prediction
and Homology Modeling of Human
Ryanodine Receptor 2
V. Bauerov-Hlinkov
1
et al.*
1
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava,
Slovakia
1. Introduction
Ryanodine receptors (RyRs) are homotetrameric intracellular calcium release channels in the
membranes of the endoplasmic (ER) and sarcoplasmic reticulum (SR) (George et al. 2005,
Meissner 2002, 2004). Each subunit consists of ~5000 amino acid residues (George et al.
2005). There are three isoforms of the ryanodine receptor: the RyR1 isoform is expressed
predominantly in skeletal muscle, the RyR2 isoform predominates in cardiac muscle, and
the RyR3 isoform is expressed in a variety of tissues (Sorrentino 1995). In the mammalian
heart, the RyR2 isoform is a principal component of the excitation-contraction (E-C)
coupling process. Action potential depolarization of the cardiac cell results in injection of
calcium ions into the cell via calcium channels (dihydropyridine receptors, DHPRs). This
small calcium influx then drives the release of calcium from intracellular calcium stores by
triggering the opening of RyR2 channels (Fabiato 1985). The released calcium causes
contraction by binding to troponin C (Ebashi and Ogawa 1988). Consequently, precise
regulation of RyR activity during heartbeat is essential to proper cardiac function.
In several cardiac diseases, such as heart failure and the genetic diseases CVPT
(catecholaminergic polymorphic ventricular tachycardia) and ARVD2 (arrhythmogenic right
ventricular dysplasia), the function of RyR is compromised. In heart failure, the release of
calcium in response to the action potential is decreased, while RyR remains more active
during the diastole (Durham et al. 2007, Yano et al. 2006). In CPVT and ARVD2, RyRs
contain mutations that lead to altered RyR activity which may result in premature calcium
release in the absence of an action potential (Durham et al. 2007, Yano et al. 2006).
In this work we present a bioinformatics analysis of the whole of human RyR2 (hRyR2) in
context with the available functional information, in order to locate individual domains for
further biochemical and structural studies. The reliability of the predictions in the N-
terminal region (Bauerova-Hlinkova et al. 2010) was verified experimentally by expressing
and characterizing the domains identified. We also describe the results of a CD-

*
J. Bauer
1
, E. Hostinov
1
, J. Gaperk
1
, K. Beck
3
, . Borko
1
, A. Faltnov
2
, A. Zahradnkov
2
and
J. evk
1
2
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia
3
School of Dentistry, Cardiff University, Heath Park, Cardiff, Wales, UK


326
spectroscopy study we carried out in order to determine the domain organization of RyR2;
this study also included an analysis of the secondary structure elements of the N-terminal
part of RyR2. Finally, we present a homology model of the N-terminal part of RyR2 which is
based on the recently determined X-ray structure of rabbit RyR1. The amino-acid sequence
identity of these two proteins is more than 80%, which suggests that predictions made from
this model will most likely be reliable. The homology model agrees with our bioinformatics
analysis and also with the results of our CD-spectroscopy study. This model should help to
locate and identify the mutations and the residues in their proximity that are responsible for
the cardiac diseases CPVT1 and ARVD2.
2. Physiological function of RyR2
Calcium release from intracellular stores is mediated by two types of calcium release
channels ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs)
(Berridge 1994). These channels are expressed in most tissues. RyRs play a primary role in
skeletal and cardiac muscle cells, where they mediate muscle contraction. In these tissues
IP3Rs play only a modulatory role. In contrast, smooth muscle, neurons and non-excitable
tissues rely on IP3R to play the primary role in calcium release, while RyRs play a
modulatory role (Berridge 1994). The study of RyRs is therefore mostly concerned with
understanding their role in the activation of skeletal and cardiac muscle contraction.
2.1 Ion permeation
The physiological role of the RyR is to allow permeation of Ca
2+
ions from the lumen of the
SR into the cytosol. Although the calcium gradient between these two compartments is high
(the diastolic free Ca
2+
concentration is only ~100 nM in the cytosol but ~12 mM in the SR
lumen (Shannon et al. 2003)), concentrations of other ions are much larger (~150 mM K
+
) or
comparable (~1 mM Mg
2+
) on both sides of the SR membrane. During the systole, >70% of
the Ca
2+
ions are released from the SR in several milliseconds. Therefore the conductance,
which determines the rate of transport, and the permeability of the channel for Ca
2+
ions,
which enables selective transport of Ca
2+
in the presence of high concentrations of other
ions, have to be sufficiently high.
The properties of RyRs have been investigated in planar bilayers by fusion of SR vesicles
(Meissner and Henderson (1987), reviewed by Meissner (2002)) and by incorporation of
purified ryanodine receptors (Lai et al. (1988), reviewed by Meissner (2002, 2004)) into
bilayer membranes. RyRs are characterised by their high conductance and relatively low ion
selectivity. Their monovalent cation conductance is very high (200700 pS), highest for the
RyR2 isoform and lowest for the RyR1 isoform, and they are half-saturated at
~1050 mM concentrations for all monovalent cations. Despite their differences in
conductance, their permeability to all monovalents is approximately equal. The channel is
6.5-fold more permeable for divalent than for monovalent ions, and their conductance (90
200 pS) (Williams 1992) increases with the size of the divalent ion. Half-saturation is
achieved at ~0.5 mM divalent ion concentration, i.e., at much lower concentrations than for
monovalent ions. These properties of the RyR channel enable effective transport of Ca
2+

ions. The large conductance ensures a sufficient transport rate, while the high calcium
affinity ensures that under normal conditions, the rate of Ca
2+
transport will be close to
maximal. The unitary Ca
2+
current under physiological conditions has been estimated to be
and Homology Modeling of Human Ryanodine Receptor 2

327
0.40.6 pA (Mejia-Alvarez et al. 1999, Kettlun et al. 2003). The permeation properties are
conferred on the RyR by the amino acids forming the pore, which are close to the C-terminal
end of the RyR (Du et al. 2001, Zhao et al. 1999), and where aa. GGIG were proposed to form
the selectivity filter (Balshaw et al. 1999, Gao et al. 2000).
2.2 Regulation by Ca
2+

Ca
2+
ions are the most important regulator of RyR activity (Fabiato 1985). They act at several
Ca
2+
binding sites, leading to activation as well as inactivation of RyR. From the
physiological point of view it is important to note that Mg
2+
ions, present at millimolar
concentrations in the cytosol and the SR lumen, are also capable of binding to all RyR Ca
2+

binding sites. The existence of two activation sites and two inactivation sites has been
proposed (Laver 2007, Laver 2009).
2.2.1 Cytosolic activation
Cytosolic Ca
2+
is the physiological activator of the RyR2 and RyR3 isoforms and contributes
to the activation of the RyR1 isoform. In the cardiac myocyte, diastolic Ca
2+
is ~ 50100 nM
(Baartscheer et al. 1998, Kagaya et al. 1995). Ca
2+
ions activate RyR channels in the
concentration range relevant for excitation-contraction coupling (0.3100 M). The
probability that RyR channels are open in the absence of other modulators increases with
increasing calcium concentration with half-activation at ~1 M (Chu et al. 1993, Coronado
et al. 1994, Gyorke et al. 1994, Meissner 2004, Zahradnikova et al. 1999).
The time course of both RyR activation (Gyorke and Fill 1993, Schiefer et al. 1995,
Zahradnikova and Zahradnik 1999, Zahradnikova et al. 1999, Zahradnikova et al. 2003) and
deactivation (Schiefer et al. 1995, Velez et al. 1997) is very rapid; the activation rate is
dependent on Ca
2+
concentration (Schiefer et al. 1995, Zahradnikova et al. 1999) while the
deactivation rate is not (Schiefer et al. 1995). The fast activation and deactivation kinetics
should allow RyRs to respond to physiological calcium signals that last only a few
milliseconds. The response of the ryanodine receptor to rapid and brief calcium elevations,
mimicking physiological stimuli, has shown that RyR has several Ca
2+
binding sites. In wild-
type RyRs, binding of at least 4 Ca
2+
ions precedes channel activation (Zahradnikova et al.
1999). RyR channels containing subunits mutated in the putative Ca
2+
binding sites are less
sensitive to activation by cytosolic Ca
2+
(Li and Chen 2001). Analysis of the calcium
dependence of RyR tetramers containing both wild-type and mutated monomers confirmed
the presence of a single Ca
2+
binding site on each of the monomers and revealed that
activation by Ca
2+
proceeds by allosteric interaction between Ca
2+
binding and channel
opening (Zahradnik et al. 2005). The cytosolic Ca
2+
binding activation site is located in the
C-terminal part of the channel (Chen et al. 1998, Li and Chen 2001). The C-terminal part of
the RyR sequence (amino acids 36615037) is capable of forming an ion channel that can be
activated by calcium (Bhat et al. 1997, Xu et al. 2000).
Due to competition between Ca
2+
and Mg
2+
ions, the apparent sensitivity of RyR2 channels
in situ to activation by calcium is decreased about 10 times by Mg
2+
binding to the activation
site (Meissner 1994). The calcium dependence of in situ RyR activity enabled elucidation of
the mechanism of the differences between the effect of Mg
2+
and Ca
2+
on RyR. While
binding of Ca
2+
to the activation site has a strong positively allosteric effect, the binding of
Mg
2+
has a weak negatively allosteric effect (Zahradnikova et al. 2010). At diastolic calcium
concentrations, more than 75% of the cytosolic activation sites are occupied by Mg
2+


328
(Zahradnikova et al. 2010). Since Mg
2+
dissociation from this site is relatively slow
(Zahradnikova et al. 2003, 2010), it limits the rate at which RyRs can respond to
physiological Ca
2+
elevations, which might play an important role in the physiological
regulation of RyRs.
2.2.2 Luminal activation
Calcium ions also affect RyR activity from the side of the SR lumen. At low cytosolic Ca
2+

concentrations, when RyR activity is low, the presence of Ca
2+
at the luminal side increases
RyR activity by prolonging channel opening if calcium current flows from the lumen to the
cytosol, i.e., by binding to the cytosolic calcium binding site (Laver 2007, Laver 2009, Xu and
Meissner 1998). Luminal Ca
2+
also affects RyR activity by binding to a luminal binding site
which may be located either on the channel or on an associated protein (Gaburjakova and
Gaburjakova 2006, Gyorke and Gyorke 1998, Gyorke et al. 2004, Qin et al. 2008). The action
of luminal Ca
2+
is complicated by the fact that the SR lumen contains a large amount of
calsequestrin, a low-affinity Ca
2+
buffer, which, in addition to its buffering effect, also
interacts with RyR and modulates its activity (see below). In planar lipid bilayers, the effect
of luminal Ca
2+
on RyR activity can be explained by a combination of direct binding to the
luminal activation site and of action at the cytosolic sites via feed-through of Ca
2+
that
passes through the channel pore to the cytosolic side of the channel (Laver 2007, 2009). Mg
2+

inhibits the luminal effect of Ca
2+
by competing with it at the luminal activation site (Laver
and Honen 2008).
2.2.3 Inactivation
Their low sensitivity to Ca
2+
-induced inactivation distinguishes RyR2 and RyR3 from the
skeletal isoform, which is half-inactivated by ~100 M Ca
2+
(Chu et al. 1993, Lamb 1993,
Smith et al. 1988). The identity of the inactivating calcium binding site is unknown. Because
of the differences between RyR1 on one hand and RyR2/RyR3 on the other, it is assumed
that the differences in sensitivity to calcium-dependent inactivation may be partially due to
the divergent region DR1, which differs between these RyR isoforms (Du et al. 2000). The
inhibitory site has low specificitythe affinity of Mg
2+
and Ca
2+
to this binding site is
similar (Gyorke and Gyorke 1998, Laver and Honen 2008, Laver et al. 1997, Xu et al. 1996).
2.2.4 Activation by ATP
ATP increases the probability that the RyR channel will be open (EC
50
= ~100 M) without
markedly affecting its calcium dependence (Xu et al. 1996). The activity of the RyR1 isoform
is potentiated more strongly than that of RyR2 (Zimanyi and Pessah 1991). Although most
ATP in the cell is present in the form of MgATP, it seems that the activating species is free
ATP
2-
(Copello et al. 2002). Other nucleosides are much less effective than ATP. ADP is a
partial agonist with a lower affinity (EC
50
= ~1 mM). Adenosine and adenine have a still
lower effect. CTP, GTP, ITP and UTP do not activate the channel at all (Meissner 2002). The
existence of the adenine ring is necessary for ATP binding, and the large effectiveness of
channel activation by ATP appears to be caused by the presence of the negatively charged
phosphate groups (Chan et al. 2000, 2003).
2.2.5 Modulation by associated proteins
Calmodulin (CaM) is a small Ca
2+
-binding protein that affects many enzymes, receptors and
channels. CaM with four bound calcium ions and apo-CaM without bound Ca
2+
ions have

329
different conformations and therefore also different effects. CaM inhibits all RyR isoforms.
Apo-CaM has a stimulatory effect on the activity of RyR1 and RyR3, but, depending on
conditions, it either does not affect or inhibits the RyR2 isoform. The effects of CaM are
mediated by its high-affinity binding to a binding site (amino acids 35833603 in RyR2) on
each of the monomers, which is conserved in all RyR isoforms. The different CaM effects on
the different isoforms are apparently due to differences in the isoforms in a region outside
the CaM binding site (Meissner 2004). The locations of bound CaM and apo-CaM on the
binding site are different (Meissner 2004), and a calcium-dependent physical relocation of
CaM on the RyR molecule has also been observed by cryoelectron microscopy (cryo-EM)
(Samso and Wagenknecht 2002). Modulation of RyR activity by calmodulin may therefore
involve conformational changes in more distant parts of the RyR protein.
FKBP (FK506-binding protein) belongs to the immunophilins, cytosolic receptors for
imunosuppresants such as rapamycin and FK506. Each RyR monomer contains a binding
site for either FKBP12 (in RyR1) or FKBP12.6 (in RyR2). The interaction of FKBP with the
channel stabilizes the protein complex and supports coordinated gating of all four subunits.
It is thought that FKBP also plays a role in coupled gating of neighbouring RyR channels
(Williams et al. 2001).
In addition to RyR channels, the SR membrane of terminal cisternae also contains the
proteins triadin and junctin (Bers 2004) which associate with RyRs from the luminal side. In
the lumen of the terminal cisterna there is also a large quantity of the protein calsequestrin
(CSQ), a low-affinity calcium-binding protein that serves as a calcium buffer and also
modulates RyR activity in a calcium-dependent manner (Bers 2004). CSQ most probably
interacts with the RyR channel through interactions with triadin and junctin, so that for
correct luminal regulation all three accessory proteins are necessary (Gyorke et al. 2004,
Terentyev et al. 2008).
2.2.6 Modulation by phosphorylation
Ryanodine receptors have several conserved regions that are putative phosphorylation sites.
Furthermore, kinases (PKA) and phosphatases (PP1 and PP2A) are directly attached to the
channel, suggesting that regulation by phosphorylation may have physiological significance
(Marx et al. 2001). The first phosphorylation site discovered on the RyR2 molecule (Witcher
et al. 1991) was S2809 (S2843 in RyR1), which can be phosphorylated by the
Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) and to some extent also by PKA.
Other kinases, such as PKG and PKC, affect RyRs as well (Takasago et al. 1991), e.g. by
changing their ability to bind the alkaloid ryanodine. The mechanisms by which
phosphorylation and dephosphorylation induce changes in RyR activity are not clearly
understood, however. An increase in RyR activity due to an increase in calcium sensitivity
(Marx and Marks 2002), as well as an increase in the rate of adaptation after a rapid calcium
increase (Valdivia et al. 1995) have both been observed after phosphorylation.
2.2.7 Interdomain interactions
Most of the mutations that affect RyR2 function in CPVT and ARVD2 are located in either of
four domains: the N-terminal domain, the central domain, the cytoplasmic I-domain, and
the transmembrane domain. This clustering, as well as the similarity between the effects of
mutations at different positions within these domains (hyperactivation of the channel and


330
increased sensitivity to agonists) led to the postulation of the interdomain hypothesis
(Yamamoto et al. 2000). It was hypothesized that there is an interaction between the N-
terminal and the central domain that stabilizes the channel in the closed state. Mutations
within these domains would then weaken this interdomain interaction (Ikemoto and
Yamamoto 2000, Yamamoto et al. 2000) and these changes might play a key role in the
regulatory mechanisms of the channel. The experimental strategy to test this hypothesis was
based on the assumption that if an interaction between two regions functions as a regulatory
mechanism, then a synthetic domain peptide with a sequence identical to that of one of the
interacting regions should destabilize the interaction and disturb RyR regulation in a way
similar to that observed in the mutations. Several domain peptides from the N-terminal and
central region of RyR1 or RyR2 (H163S195, L590C609 and L601C620, L2442P2477 and
G2460P2495, D2380A2411) were indeed able to activate RyR and increase its sensitivity to
agonists (El-Hayek et al. 1999, Faltinova et al. 2011, Tateishi et al. 2009, Yamamoto and
Ikemoto 2002, Yang et al. 2006). The group of Yamamoto and Ikemoto further postulated,
based results of George et al. (2004), that the interaction between the I-domain and another,
undefined domain also plays a role in stabilizing the closed state of RyR2. A domain peptide
from the I-domain (P4090E4123) did indeed activate RyR2 (Tateishi et al. 2009).
Furthermore, mutations in the N-terminal and central domains destabilized the interaction
of RyR2 with its regulator calmodulin (Ono et al. 2010). Two RyR-stabilizing drugs that act
on the central (K201) and N-terminal domains (dantrolene) antagonized the effect of the
central and N-terminal mutations, respectively, and restored calmodulin binding (Ono et al.
2010, Xu et al. 2010). Transmission between the interdomain interaction, calmodulin
binding, and channel opening is believed to be mediated by a calmodulin-like domain
(residues 40644210 in RyR1; (Xiong et al. 2006)) and its interaction with the calmodulin
binding domain of RyR (Ono et al. 2010). The effect of CPVT and ARVD2 mutations on the
extent of interdomain interaction may be manifested as a change in the intrinsic opening
tendency of the channel or in the allosteric effect between calcium binding and channel
opening (Zahradnikova et al. 2010).
2.3 Excitation-contraction coupling
The RyRs are located at tubulo-reticular junctions, special calcium release sites, where they
face the adjacent calcium channels (DHPRs) of the plasma membrane. The release sites in
both skeletal and cardiac muscle contain clusters of RyRs and DHPRs, and their structures
are quite similar. However, DHPRs and RyRs are positioned very precisely adjacent to one
another in skeletal muscle but not in cardiac muscle (Protasi 2002). Therefore, in contrast to
skeletal muscle, where RyR1 is activated by conformational changes in the DHPR protein
(Rios and Brum 1987, Rios et al. 1993), in cardiac cells RyRs are activated by calcium ions
that flow into the dyadic space during single-channel openings of DHPRs (Fabiato 1985).
Calcium sparks, calcium release events of individual calcium release units (Cheng et al.
1993), raise local cytosolic Ca
2+
concentrations by 200 nM (Cannell et al. 1994, Cannell et al.
1995, Cheng et al. 1993, Lopez-Lopez et al. 1995). They can be discerned when release
probability is low (Cheng et al. 1993). Although the probability of spontaneous sparks in a
rat ventricular myocyte is low (about 100 s
-1
) (Cheng et al. 1993), openings of L-type Ca
2+

channels greatly increase this probability during a voltage step depolarization (Cannell et al.
1995, Lopez-Lopez et al. 1995). Ca
2+
sparks are stereotypical, i.e., their amplitudes and

331
spatio-temporal properties appear to be independent of their trigger signal (Cannell et al.
1995, Lopez-Lopez et al. 1995). This indicates that the amplitude and time course of a Ca
2+

spark is largely governed by the properties of the participating RyRs. Thus, Ca
2+
sparks can
be considered to be the elementary Ca
2+
release events underlying E-C coupling, and
gradation of calcium release in response to I
Ca
can be explained by the summation of
variable numbers of Ca
2+
sparks being activated (Cheng et al. 1996).
Under normal conditions, Ca
2+
sparks are unable to activate additional Ca
2+
sparks in
adjacent regions, although the local free Ca
2+
concentration associated with a Ca
2+
spark is
much larger than the global increase in free Ca
2+
concentration produced by Ca
2+
current
activation ((Cannell et al. 1995), but see Parker et al. (1996)). This observation can be
explained by the fact that SR Ca
2+
release channels are situated very close to the L-type Ca
2+

channel (the local control model; (Stern 1992)), where they sense the > 100-fold increase in
local free Ca
2+
concentration upon opening of a nearby L-type Ca
2+
channel. The sensitivity
of this local control is most clearly seen in the evidence that a single DHPR channel opening
can elicit a Ca
2+
spark (Cannell et al. 1995, Lopez-Lopez et al. 1995, Santana et al. 1996). A
detailed analysis revealed that the probability of Ca
2+
activation depends on the square of
the single sarcolemmal Ca
2+
channel current and on the square of the local free Ca
2+

concentration (Santana et al. 1996) and that it is quite sensitive to the duration of the DPHR
openings (Zahradnikova et al. 1999). Theoretical calculations (Cannell and Soeller 1997) as
well as experimental data (Zahradnikova et al. 1999) suggest that RyRs can respond rapidly
to calcium influx via DHPRs; the responsiveness of RyRs in situ depends on the geometrical
arrangement of channels in the narrow dyadic space, in which high Ca
2+
levels rapidly
develop near the SR membrane. Such high Ca
2+
levels lead to a rapid rate of binding of
calcium to the RyR calcium sensitive sites, thereby reducing the latency between DHPR
opening and SR calcium release to ~1 ms, in correspondence with the experimentally
observed DHPR mean open times. However, direct measurements of the latency between
long (~20-ms) calcium channel openings and spark activation (Wang et al. 2001) indicated a
much longer latency (~7 ms). Since the binding of Mg
2+
(Zahradnikova et al. 2010,
Zahradnikova et al. 2003) has not been included in the model (Cannell and Soeller 1997), it
may be inferred that only RyR channels that do not have Mg
2+
bound are able to respond
rapidly to DHPR openings (Zahradnikova et al. 2010, Zahradnikova et al. 2003).
It is not clear how many RyR channels contribute to a Ca
2+
spark. While there are 5-9 RyRs
per DHPR in cardiac myocytes, individual Ca
2+
sparks may reflect activation of up to 20
RyRs (Bridge et al. 1999, Lukyanenko et al. 2000). This means that the majority of RyRs are
activated by neighbouring RyRs and not by adjacent DHPR channels. A quantitative model
of spark activation based on the allosteric action of Ca
2+
and Mg
2+
on RyR2 opening
(Zahradnikova et al. 2010) predicts the opening of 18 RyR2 channels per spark, in
agreement with experimental findings (Wang et al. 2001).
3. Structure of ryanodine receptors
3.1 Overall structural features determined by cryoelectron microscopy (cryo-EM)
RyR channels are the largest ion channels known so far, which makes structural studies of
them very challenging. So far, the structure of a whole RyR has been determined only by
cryo-EM. Most of these studies, including sub-nanometer EM (Samso et al. 2009,


332
Serysheva et al. 2008) have focused on the skeletal RyR1 isoform, but some studies have
been performed on RyR2 (Liu et al. 2002, Sharma et al. 1998) and RyR3 (Liu et al. 2001,
Sharma et al. 2000) as well. In agreement with the high sequence homology of RyRs,
which reaches ~65%, EM structures of all three isoforms are very similar (Wagenknecht
and Samso 2002). The RyR2 (Sharma et al. 1998) and RyR3 (Sharma et al. 2000) isoforms
differ slightly from the RyR1 isoform in several structural domains (called divergent
regions) that map to segments of reduced homology between the RyR isoforms (Zhang et
al. 2003), though the overall structure of the RyR isoforms is very well conserved
(Wagenknecht and Samso 2002). The complete channel is made from a combination of
four monomers to yield a tetramer with a fourfold axis of symmetry. The divergent
regions have been suggested to play a role in calcium-dependent inactivation (DR1) (Du
and Maclennan 1999), in signal transmission between DHPR and RyR1 (DR2) (Perez et al.
2003), and in conformational changes of the RyR and RyR-RyR interactions (DR3) (Zhang
et al. 2003).
Each subunit of the RyR homotetramer consists of two main parts: a cytoplasmic part and a
transmembrane part. The cytoplasmic part of the whole receptor, also called the foot, is
very large (280 280 120 ) and interacts with many modulators which affect channel
gating (Yano et al. 2006). It is composed of several structural segments: the clamp and the
handle at the perimeter, the central rim surrounding the putative pore, and the column
connecting the cytoplasmic and transmembrane parts (Fig. 1). These segments have been
further subdivided into 15 subdomains (Lanner et al. 2010). The clamps are located at the
corners of the cytoplasmic part and are likely to participate in intermolecular interactions
with neighboring RyR molecules and other RyR modulators. They undergo large changes
during the opening and closing of the channel (Orlova et al. 1996, Samso et al. 2009,
Serysheva et al. 1999). Like the cytoplasmic domain, the transmembrane part undergoes
large structural changes during the opening and closing of the RyR2 channel (Orlova et al.
1996, Samso et al. 2009, Serysheva et al. 1999).
3.2 Bioinformatics domain prediction
To find the putative individual structural entities of hRyR2, we analyzed the whole hRyR2
amino acid sequence using the PFAM domain database (Finn et al. 2008). Fourteen probable
domains were found in the hRyR2 monomer. Eight of them are located in the N-terminal
region (residues 1~1561) and were identified as Ins145_P3_rec, MIR, RIH, SPRY and two
RyR domains. The central region (residues 15623000) contains the RIH domain and two
RyR domains. The C-terminal part (residues 30014995) contains an RIH associated domain,
a RR_TM4-6 domain and an Ion_Trans domain, Fig. 2. The beginnings and ends of each
domain are numbered according to the PFAM search results. Mutations of specific residues
involved in ARVD2 and CPVT1 are shown (Yano et al. 2006; www.fsm.it/cardmoc). LIZ1-3
(amino acid residues 554585, 16031631, and 30033039) represent leucine-isoleucine zipper
areas. The proposed binding partners, SPI, PR130, mKAP, PP1, PP2A, PKA, D, K201, FKBP,
and CaM represent spinophilin, protein 130, muscle specific kinase anchoring protein,
protein phosphatases 1 and 2A, protein kinase A, dantrolene, 1,4-benzothiazepine derivative
K201 (JTV519), FK-binding protein, and calmodulin, respectively. Binding partners for
hRyR2 and their positions were adapted from Wang et al. (2011), Yamamoto et al. (2008),
and Yano et al. (2006).

333

Fig. 1. A cryo-EM density map of RyR1 (accession no. 1274, http://www.ebi.ac.uk/emdb-
srv/atlas/1274_summary.html) in the in closed state. A. Cytopasmic view. B. Side view. The
cytoplasmic (clamp, handle, central rim and column) and transmembrane regions (TM) are
indicated.

Fig. 2. Analysis of the human cardiac ryanodine receptor using the PFAM database. The
position and type of mutations resulting in CVPT or ARVD2, as reported at
www.fsm.it/cardmoc/, are indicated above the sequence regions.


334
3.3 X-ray analysis
To date, X-ray structures have been determined for several N-terminal fragments of rabbit
RyR1 (residues ~12210 (Amador et al. 2009, Lobo and Van Petegem 2009); residues 12532
(Tung et al. 2010)); and murine RyR2 (residues 12217, wt and mutants A77V and V186M)
(Lobo and Van Petegem 2009). The overall structures of all isoforms, including those
containing mutations, are very similar (superposition results in r.m.s.d. of 0.69 for 150 C

atoms), Fig. 3, which is not surprising due to their close sequence homology and
physiological function. The longest fragment, residues 12532, is composed of three
structural domains, which have been designated as A (1205), B (206394) and C (395532)
(Tung et al. 2010). Domains B and C are homologous respectively with the -trefoil and -
helical domains of the IP3R binding core. Domain A is homologous with the IP3 binding
suppressor domain of IP3R (Yuchi and Van Petegem 2011). The central motif of domains A
and B is a -trefoil core consisting of 12 -strands which are held together by hydrophobic
interactions. In the A domain, a 10-residue -helix is inserted between strands 4 and 5
(Lobo and Van Petegem 2009). Domain C consists of five -helices. Most of the secondary
structure elements are connected by flexible loops, which were proposed to be located at the
interfaces with other RyR domains or at the interfaces with proteins interacting with RyR
(Tung et al. 2010, Yano et al. 2006). The X-ray crystal structures allowed the precise mapping
of several mutations which are associated with CPVT and ARVD2 (Lobo and Van Petegem
2009), as well as the homologous mutations in RyR1 which are responsible for malignant
hyperthermia (MH) and central core disease (CCD) (Tung et al. 2010).

Fig. 3. Comparison of X-ray structures of the N-terminal domains of RyR1 (PDB ID 2XOA
green, 3HSM pink, 3ILA magenta) and RyR2 (PDB ID 3IM5 blue, 3IM6 yellow, 3IM7
purple). The superposition was performed using the program Multiprot (Shatsky et al.
2004), through 150 C
atoms with a r.m.s.d. of 0.69 . Arrows indicate flexible loops.


335
3.4 Relationships between domain structure, 3D structure and RyR function
3.4.1 The Ins145_P3_rec domain
The Ins145_P3_rec domain is found in RyRs and IP3Rs (Ponting C. P. 2000). In IP3R, it
participates in forming the ligand binding suppressor region (Bosanac et al. 2005). In the
structure of RyR1, it is equivalent to the first N-terminal domain A (Tung et al. 2010). The
PFAM prediction assigned this domain to residues 9223, while in the RyR1 structure it
corresponds to the equivalent RyR2 amino acids 12228. This domain contains two closely
spaced clusters of mutations associated with CPVT and ARVD in the region of residues 62
81 and 164189 (http://www.fsm.it/cardmoc), which belong to the first mutation cluster
CPVT-I (George et al. 2007). The domain consists of 12 -strands, arranged into a -trefoil
motif (see below). Superposing the ligand-binding suppressor domain of the IP3R (PDB ID
1XZZ; (Serysheva et al. 2008)) and the X-ray structure of the N-terminal portion of RyR1 into
the cryo-EM density map of RyR1 (Tung et al. 2010) indicates that this domain should lie in
the clamp or on the central rim of the ion channel, respectively.
3.4.2 The MIR domain
MIR domains are found in a number of proteins (George et al. 2007, Hamada et al. 1996,
Strahl-Bolsinger and Scheinost 1999), including IP3Rs (Bosanac et al. 2005, Bosanac et al.
2002), and RyRs (Amador et al. 2009, Lobo and Van Petegem 2009, Ponting 2000, Tung et al.
2010). They usually consist of several ~50-residue MIR motifs with a -trefoil fold (Murzin et
al. 1992), and form -barrel structures with hairpin triplets and internal pseudo-threefold
symmetry (Bosanac et al. 2005). In RyR1, the MIR domain is equivalent to the second N-
terminal domain, domain B (Tung et al. 2010). In PMT1 mannosyltransferases, MIR motifs
are located in the luminal loops of the enzyme and are essential for transferase activity
(Stahl-Bolsinger et al., 1999). In IP3R, the first two of the -trefoil motifs were found to
belong to the suppressor region (Ins145_P3_rec, (Bosanac et al. 2002), see above), while the
latter two (parts of the MIR domain) belong to the ligand binding region (Bosanac et al.
2005). Similar -trefoil motifs were predicted to be present in the N-terminal region of the
RyR1 isoform (Bosanac et al. 2002), and were later found in its crystal structure (Amador et
al. 2009), although the sequence similarity between IP3R and RyR is relatively low. PFAM
predicted this domain to lie between residues 226406 of RyR2, and in the RyR1 structure it
corresponds to the equivalent RyR2 amino acids 228411. This region contains a large
number of CPVT/ARVD2 mutations (Fig. 2) (http://www.fsm.it/cardmoc/), which belong
to the first mutation cluster CPVT-I (George et al. 2007). Docking of the ligand-binding
domain of IP3R (PDB ID 1N4K, (Serysheva et al. 2005); PDB ID 1XZZ and PDB ID 1N4K,
(Serysheva et al. 2008)) into the cryo-EM structure of RyR1 predicts that this domain will lie
in the clamp region while doing a similar docking using the X-ray structure of the N-
terminal sequence of RyR1 (Tung et al. 2010) indicates that the MIR domain should lie on
the central rim.
3.4.3 The RIH domains
Two RIH domains were found in both RyRs and IP3Rs. The X-ray structure of a major part
of the first RIH domain of both IP3R (Bosanac et al. 2002) and RyR1 (Tung et al. 2010) has
been determined. In the case of RyR1, this corresponds to domain C and the structure
extends out to residue 532, which corresponds to residue 543 in hRyR2. Structurally, RIH is
composed of -helices. In IP3R, this domain forms the binding site for inositol 1,4,5-


336
trisphosphate (Bosanac et al. 2002). By superposing the ligand suppressor domain (PDB ID
1XZZ) and the ligand binding core (PDB ID 1N4K) on the N-terminal part of RyR1 (PDB ID
2XAO), it was proposed that all three domains of IP3R, i.e. Ins145_P3_rec, MIR and RIH,
interact together, as predicted by Chan et al. (2007), and are arranged similarly as in the N-
terminal part of RyR1 (Yuchi and Van Petegem 2011). The PFAM prediction placed this
domain between residues 451655, while in the RyR1 structure its beginning corresponds to
the equivalent RyR2 amino acid 410. In RyR2, the RIH domain was reported to contain a
leucineisoleucine zipper between amino acid residues 554 and 585 that mediates binding of
the phosphatase PP1 via the spinophilin targeting protein (Marx et al. 2001). This domain
was also proposed to contain the binding site for dantrolene (residue 626 in hRyR2), and it
contains several of the CPVT/ARVD2 mutations (http://www.fsm.it/cardmoc/), which
belong to the first mutation cluster CPVT-I (George et al. 2007). It was located in the clamp
region by cryo-EM (Wang et al. 2011). However, docking of the X-ray structure of the N-
terminal sequence of RyR1 into the cryo-EM structure of RyR1 predicts that this domain lies
in the central rim (Tung et al. 2010).
PFAM predicted that the second RIH domain lies between residues 2121-2332. This domain
and its C-terminally adjacent region contain the central cluster of CPVT/ARVD mutations
(CPVT-II, (George et al. 2007), http://www.fsm.it/cardmoc/) and is flanked by putative
binding sites for the protein FKBP 12.6. Cryo-EM places this region in the clamp (Liu et al.
2005, Wang et al. 2011).
3.4.4 The SPRY domains
The SPRY domain (sp1A kinase and the ryanodine receptors) (Ponting et al. 1997) structurally
consists of several antiparallel -strands connected with flexible loops. The precise function of
the SPRY domain (and the related B30.2 domain) is unknown; however, it is believed to act as
a proteinprotein interaction module capable of binding multiple targets by recognizing the
conformation of a partner protein rather than a consensus sequence motif (Woo et al. 2006, Yao
et al. 2006). The B30.2/SPRY domain has been identified in numerous and diverse proteins
across bacterial and eukaryotic species (e.g. pyrin/marenostrin and other butyrophilin-like
homologues, ryanodine receptors and midin1), including over 150 proteins in humans
(Rhodes et al. 2005), suggesting that the specific function of the B30.2/SPRY domain within a
given protein may heavily rely on the other domains in their neighbourhood (Kleiber and
Singh 2009). PFAM predicted that RyR2 contains three SPRY domains, corresponding to
residues 670808, 10981221, and 14331561. The first two domains contain three
CPVT/ARVD mutations: R739H, T1107M and A1136V (Medeiros-Domingo et al. 2009), Fig. 2,
which lie outside of the four mutation clusters.
3.4.5 The RyR domains
Four copies of the RyR domain are present in the ryanodine receptor, of which two belong
to the N-terminal and two to the central regions. The function of this domain is unknown
(Ponting 2000). In the second RyR domain, two isolated CPVT mutations, which lie outside
of the four mutation clusters, have been found to date: R1013Q and R1051P, Fig. 2
(Marjamaa et al. 2009, Medeiros-Domingo et al. 2009).
3.4.6 The RIH_associated and RR_TM4-6 domains
According to PFAM, the RIH_associated domain should lie between residues 34653960.
This domain contains the calmodulin binding site, which was localized to the column region

337
of RyR according to cryo-EM (Samso and Wagenknecht 2002). The adjacent RR_TM4-6
domain was predicted to lie between residues 43334599 by PFAM. It contains the divergent
region DR1 (Liu et al. 2002), the putative calcium sensor that is responsible for the
physiological activation of RyR2 (Chen et al. 1998, Li and Chen 2001), and also the
calmodulin-like domain (Xiong et al. 2006). The end of the RIH_associated domain together
with the RR_TM4-6 domain (aa. 37224610) were identified as a separate functional domain
(called the I-domain) (George et al. 2004), which contains a third cluster of CPVT/ARVD
mutations (CPVT-III, (George et al. 2007), http://www.fsm.it/cardmoc/); cryo-EM locates
this domain in the column of RyR1 (Wang et al. 2011).
3.4.7 The Ion_Trans domain
The Ion_Trans (ion transport) domain covers the transmembrane region of the ryanodine
receptor. This domain is found in most voltage-dependent ion channels as well as in RyRs
and IP3Rs. The domain usually has six transmembrane helices, the final two of which flank
a loop that determines ion selectivity (Unnerstale et al. 2009). The tetrameric ion channels
(potassium channels, IP3Rs and RyRs) contain one Ion_Trans domain per monomer, while
the sodium and calcium channels contain four Ion_Trans domain repeats. This domain is
located in the transmembrane region, embedded in the membrane of the SR, Fig. 1A,B, and
contains the ion conducting pore. It has been proposed that the pore includes the
GVRAGGGIGD amino acid sequence (Du et al. 2001, Zhao et al. 1999), where the amino
acids GGIG were proposed to form a selectivity filter (Balshaw et al. 1999, Gao et al. 2000).
The fourth cluster of CPVT/ARVD2 mutations (CPVT-IV, (George et al. 2007),
http://www.fsm.it/cardmoc/) occurs in this domain and in the flanking regions on both its
sides.
3.4.8 Regions without a known domain structure
Two of the three regions of isoform sequence diversity (DR2 and DR3; (Perez et al. 2003,
Zhang et al. 2003)) are located outside of the PFAM-predicted domains. DR2 is located
between the second and the third SPRY domains (residues 1353-1397, Liu et al. 2004), while
DR3 (residues 1852-1890) is located between SPRY3 and RIH2. Both DR2 and DR3 are found
in the clamp region of RyR1 (Wang et al. 2011). DR3 contains two isolated CPVT/ARVD2
mutations, G1885E and G1886S (Milting et al. 2006), which lie outside of the four mutation
clusters.
The second and third leucine-isoleucine zipper (aa. 16041644 and 30043041), that were
found to bind PP2A and PKA with the help of the adapter proteins PR130 and mAKAP,
respectively (Marx et al. 2001), are also located outside the PFAM-predicted domains. The
location of these motifs in the 3D structure of the RyR2 is unknown.
4. Cloning, expression and characterization of predicted N-terminal hRyR2
domains
In our previous work we concentrated on the production and characterization of
recombinant N-terminal domains of hRyR2 (residues 1759) in Escherichia coli expression
systems ((Bauerova-Hlinkova et al. 2010); unpublished results). Based on the bioinformatics
analysis described above, we assumed that the predicted domains would form individual
entities and might behave as stable proteins. We designed several constructs covering the


338
predicted first three N-terminal domains (Ins145_P3_rec, MIR, RIH) with various starting
and terminating residues, Table 1, taking into consideration the predicted secondary
structure elements and the known structure of the related IP3R domains. All fragments were
designed not to disrupt the predicted secondary structure elements, Fig. 6. In this study we
obtained three authentic recombinant hRyR2 fragments with good expression yields and
solubility: 1606 (involves first three putative N-terminal domains), 391606 and 409606
(involves the core of the predicted RIH domain) and several hRyR2 fragments expressed
with a fusion partner, either thioredoxin or Nus A protein, Table 1.

hRyR2 fragment Calculated M
w
10
3
Protein Expression
1247.His
6
27.9 ++
1606.His
6
68.6 ++
391606.His
6
25.2 ++
409606.His
6
23.2 +++
Nus.1606 128.6 ++
Nus.230606 104.0 ++
Trx.384606.His
6
43.3 +++
Trx.391606.His
6
42.5 +++
Trx.409606.His
6
40.5 +++
Table 1. Designed fragments of the N-terminal part of hRyR2 with good expression and
solubility. The fragment 1247.His6 contains the Ins145_P3_rec domain. The longest N-
terminal hRyR2 fragment 1606.His6 involves all three putative N-terminal domains. The
hRyR fragments 384606, 391606 and 409606 involve the core of the RIH domain. To
improve solubility, some fragments were expressed with fusion partners Nus A protein
and thioredoxin. Quantification of expression: ++ 15 mg/g expressed cells, +++ more than
5 mg/g expressed cells. The amount of the protein was determined after IMAC purification
(Bauerova-Hlinkova et al. 2010).
The folding and thermal stability of these expressed fragments was assessed by circular
dichroism (CD) spectroscopy (Fig. 4) and a thermofluor shift assay (Fig. 5). The secondary
structure content of the fragments was derived from the CD spectra using the CDsstr
algorithm (Johnson 1999) with an extensive set of reference databases (Whitmore and
Wallace 2004). For the longest fragment, 1606, the amount of -helices and -strands
resulted in ca. 23 and 29%, respectively (Fig. 4A; (Bauerova-Hlinkova et al. 2010). For the C-
terminal domain covering residues 409 to 606 (Fig. 4B), a higher degree of -helicity (ca.
50%) was found. This value is lower than expected from the model for this region (62%, see
section 4.1.), which indicates that the expected N-terminal helix might be only partially
folded. Such disorder within the terminal regions is frequently observed in NMR and X-ray
derived structures of protein fragments. The spectra of the thioredoxin fusion protein
fragments 384606 (Fig. 4C) and 409606 (Fig. 4D) indicated -helix and -strand contents of
ca. 40 and 10%, respectively.
With temperature increasing up to 35C, only small changes of the CD signal could be
observed for fragment 1606 (Fig. 5A), but further heating resulted in irreversible
precipitation under our experimental conditions (Bauerova-Hlinkova et al. 2010). A similar
behaviour was observed for the fragment 409606, which was stable up to 37C (Fig. 4B) and
started to precipitate at T > 42C (data not shown). When the fragment 409606 was fused

339
with thioredoxin A, its CD signal indicated a gradual loss of -helicity (ca. 25% at 95C) that
was fully recovered upon cooling (data not shown).

Fig. 4. Far-UV CD spectra of recombinant hRyR2 fragments 1606 (A) and 409-606 (B), and
as fusion proteins with thioredoxin A at the N-terminus Trx-384-606 (C) and Trx-409-606
(D). Spectra were recorded in an 0.02-cm cell at 4C (solid line) and 37C

(dashed line; 35C
for hRyR2 1606). Samples were dialyzed into 100 mM NaF, 20 mM Tris SO
4
pH 7.5 or 8.0,
including either 0.1% Tween-20 (A-C) or sulfobetaine SB14 (D). Deconvolution of the 4C
spectra using the CDsstr algorithm as implemented in Dichroweb with various reference
databases ((Whitmore and Wallace 2004); http://dichroweb.cryst.bbk.ac.uk/) results in the
amount of -helix and -strand shown at the top right of each panel.
The results of the thermofluor shift assay, performed with the longest hRyR2 fragment 1
606, were in good agreement with the temperature dependence of the circular dichroism
spectra. The thermal stability of the fragment was tested in a wide range of buffers (Tris,
Hepes, MES, citrate, Na/K phosphate, Bicine, Tricine; pH range 5.09.0), Fig. 5, and in the
presence or absence of 1 and 5 mM Ca
2+
, Mg
2+
, ATP and 520% glycerol. The fragment is the
most stable in neutral-basic pH (7.08.0) with a T
m
of ~45C, Fig. 5B, C. The presence of Ca
2+


340
or ATP did not change the T
m
significantly, suggesting that the fragment does not contain
binding sites for these ligands.

Fig. 5. The first derivative of the thermal denaturation curve of recombinant 1606 hRyR2
fragment obtained by thermofluor shift assay, measured in 200 mM Na-citrate, pH 5.5 (A),
and 200 mM Na-phosphate, pH 7.5 (B). C. T
m
of the recombinant hRyR2 fragment 1-606
obtained by the thermofluor shift assay in different buffers and pH (1 K-phosphate 5.0; 2
Na-phosphate 5.5; 3 Na-citrate 5.5; 4 MES 5.8; 5 K-phosphate 6.0; 6 MES 6.2; 7 Na-
phosphate 6.5; 8 Na-cacodylate 6.5; 9 MES 6.5; 10 K-phosphate 7.0; 11 HEPES 7.0; 12
Na-acetate 7.3; 13 Na-phosphate 7.5; 14 Tris 7.5; 15 Imidazol 8.0; 16 HEPES 8.0; 17
Tris 8.0; 18 Bicine 8.0; 19 Tris 8.5; 20 Bicine 9.0). The conditions under which RyR2 1
606 was the most stable are in red. For each measurement 8 g of protein were used.
4.1 Model structure of the N-terminal part of hRyR2
The amino-acid sequence of the recently determined structure of the N-terminal region of
rabbit RyR1 (PDB ID 2XOA (Tung et al. 2010)) is 63% identical and 77% similar to the
corresponding sequence of hRyR2 (similarity is here defined as having a Gonnet Pam250
matrix score > 0.5 as determined by ClustalX 2.1). The 2XOA structure therefore represents
an excellent template for constructing a homology model of the N-terminal region of hRyR2.
The structure covers residues 12543 of the hRyR2 sequence. The homology model was
constructed using Modeller 9v8 (Sali and Blundell 1993). The hRyR2 sequence was first
aligned with the template structure using the alignment.align2d command of Modeller and
then manually edited to improve the alignment (Fig. 6). This alignment was used to build a
homology model using the automodel class. Residues 90107 of the human sequence, which
had been disordered in the template structure, were constrained to form an -helix in
accordance with the secondary structure predictions, see below. The structure was
thoroughly refined using automodel.library_schedule = autosched.slow and
automodel.max_var_iterations = 300 for the initial optimization step, which was then
followed by molecular dynamics refinement using automodel.md_level = refine.slow. The
whole process was repeated twice. The refinement target function (objective function)
minimized the geometry restraints and Charmm energy terms enforcing proper
stereochemistry (described in (Sali and Blundell 1993)).

341

Fig. 6. The sequence alignment of hRyR2 and rabbit RyR1 (PDB ID 2XOA) sequences which
was used as a template for the homology modeling of the hRyR2 tertiary structure. The
alignment was performed using the alignment.align2d command of Modeller 9v8 (Sali and
Blundell 1993). The secondary structure elements of hRyR2 (-helices and -strands are
shown as red bars and blue arrows, respectively) were predicted by Jpred (Cole et al. 2008).
The numbering of predicted secondary structure elements of hRyR2 corresponds to those
found in the RyR1 template structure (-helices and -strands are shown as pink bars and
light blue arrows, respectively). The alignment covers residues 1543 of the human RyR2
protein. Residues in grey are missing in the RyR1 structure and correspond to flexible loops.
Identical residues (~64%) in both sequences are labelled by asterisks.


342
Twenty trial structures were generated and the best one was chosen by first discarding all
those with unreasonable geometry in the loop regions, and then selecting the one with the
lowest objective function score. The loops in this structure were then refined using the
functions found in the Modeller loopmodel class (described in Fiser et al. (2000)). This was
done in two stages. First, the loop containing helix 1A (see below) was refined to give five
different positions. Second, for each of these positions an additional five structures were
generated with different positions for all of the loops. The final structure was selected based
on the lowest objective function. The final structure was inserted into the RyR1 crystal
packing arrangement to check for possible clashes with neighbouring molecules, which
might indicate unlikely loop conformations; none were found. The final model is shown in
Fig. 7A,B.
The hRyR2 model contains three domains, aa. 12219 (Ins145_P3_rec), 228408 (MIR) and
411543 (RIH; residue numbers refer to the hRyR2), analogous to those of the template
structure (Tung et al. 2010). The RyR1 template structure and hRyR2 homology model are in
agreement with the PFAM predictions shown in Fig. 2; however, the beginning of the third
domain, RIH, is shifted by about 40 aa. residues towards the N-terminus.
With a few exceptions, the hRyR2 homology model (Fig. 7) confirmed the secondary structure
predictions, nearly all of the -strands (22 out of 27) were found in the predicted positions in
the template structure, although with a minor shift of one to three residues, Fig. 6.

Fig. 7. (A) hRyR2 homology model constructed by Modeller 9v8 (Sali and Blundell 1993)
using the alignment with rabbit RyR1, shown in Fig. 6. Loops which were disordered in the
template structure and the modelled 1A-helix are in orange. Residues which cause
physiological dysfunction of hRyR2 when mutated are indicated. (B) The RyR2 homology
model coloured according to the domains predicted using the PFAM database. The colour
scheme is the same as in Fig. 2; Ins145_P3_rec is green, MIR is red, and RIH is yellow. The
grey -helix, 2 (aa. 409436), according to the PFAM results, precedes the RIH domain, but
is not part of the MIR domain.
The beginning of the first -strand (1A; aa. 1116) was missing in the template structure so
that its presence could not be verified. The short -strands 5A, 9A, and 13A were not
present in the RyR1 structure. 23 was present in the template and the modelled structure
but was not predicted by JPred. Three long -helices (2, 3, and 6) were correctly

343
predicted by JPred with small variations at the beginnings or the ends in the model. In the
region of the predicted helix 4, two shorter -helices were modelled which are separated
from each other only by a two-residue turn, as found in the template structure. Neither the
shortest -helix (aa. 325328) nor four out of the five 3
10
helices were predicted.
The first predicted -helix (1) deserves additional consideration. In comparison to RyR1, the
RyR2 sequence contains a 12-residue insertion, Fig. 6, suggesting that the structure of this
region differs in these two proteins. In hRyR2, helix 1 was predicted to be 33 residues long
(residues 75107). However, the Ins145_P3_rec domain, which includes the helix 1, was
determined independently three times in RyR2 and contains only the first ten residues of this
helix, followed by a gap (Lobo and Van Petegem 2009). This indicates that the predicted helix
1 cannot span the whole range. Instead, most likely, this region forms a helix-turn-helix motif
(aa. 75110), as found in the structure of the IP3R ligand binding suppressor domain (Bosanac
et al. 2005). The motif has to be rather flexible to explain its absence in the previously solved
RyR structures as well as the conformational flexibility predicted by (Bosanac et al. 2005).
Inclusion of an 18-residue 1A, separated from the helix 1 observed in the template structure
by a six-residue linker, resulted in five different conformations, which shows that the
modelled motif has sufficient mobility to explain its absence in the solved structures. Most
likely, this motif will be stabilized in the whole RyR2 protein by interaction with a binding
partner (another RyR2 domain or an interacting ligand).
Between the helix 1A and the long disordered loop containing residues 464477, there is a
very large cavity, which suggests the existence of a large binding pocket. The helix 1A and
the loop 464477, which sit atop of this cavity, both contain several aromatic residues (W98,
F100, H469, H472). The presence of the aromatic residues in the surface, three of which are
exposed to the cavity (F100, H469, H472) may indicate protein-protein binding events with a
putative ligand. The P466A mutant, located at the beginning of the loop, is known to
substantially disrupt the proper physiological function of RyR2, causing syncope (Tester et
al. 2005). Proline residues have lower conformational flexibility than other residues, so the
most likely reason for the importance of P466 would be a necessity to decrease the flexibility
of the loop containing residues 464477. In addition to the P466A mutant, two other mutants
in this area are also known to cause RyR2 dysfunction: A77V, causing CPVT and ARVD2
(d'Amati et al. 2005), and V186M, causing CPVT (Tester et al. 2006). All three mutations in
this area induce only small changes in the surface shape of the protein. Taken together, all
this implies that this part of the structure might be involved in the binding of RyR2 to other
interacting proteins or its own domains. This is consistent with the results seen in RyR1,
where the docking of the first three N-terminal domains into the full-length cryo-EM density
map revealed that the loop involving P455 (P466 in hRyR2) as well as the beginning of helix
4 belong to interface 6 (Tung et al. 2010).
The reliability of the hRyR2 homology model is further confirmed by the CD-spectroscopy
of hRyR2 fragment 1606, Fig. 4A, which revealed 23% -helices and 29% -strands in the
fragment. This is in good agreement with the hRyR2 model structure, in which the content
of -helices and -strands was 23% and 24%, respectively.
5. Conclusion
In this work, bioinformatics analysis of the whole human RyR2 is presented. The analysis
shows that the protein is composed of 14 domains. We were concerned particularly with the


344
first three N-terminal domains of the protein (Ins145_P3_rec, MIR, RIH). Verification that
the domains identified can behave as separate, independent protein units was provided by
their successful expression in E. coli and subsequent characterization. CD-spectroscopy was
used to determine the domain organization and to identify the secondary structure elements
of the N-terminal part of hRyR2. The amino acid sequence identity of hRyR2 with that of
rabbit RyR1, the X-ray structure of which is known, is higher than 60%, which allowed the
construction of a reliable homology model of the N-terminal part of hRyR2. Its reliability
was further strengthened by its conformity with the bioinformatics analysis and the CD-
spectroscopy study. This model should allow a clearer insight to be gained into the possible
influence of mutations on the cardiac diseases CPVT1 and ARVD2.
6. Acknowledgment
This work was supported by VEGA grants 2/0131/10 and 2/0190/10 and by APVV-0628-10
and APVV-0721-10. VB would like to thank Dr. Elena Hlinkov for encouragement and
support during the writing of the chapter.
7. References
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structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a
disease-associated mutation "hot spot" loop. Proc Natl Acad Sci U S A 106: 11040-
11044.
Baartscheer A, Schumacher CA, Fiolet JW. 1998. Cytoplasmic sodium, calcium and free
energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes. J Mol Cell
Cardiol 30: 2437-2447.
Balshaw D, Gao L, Meissner G. 1999. Luminal loop of the ryanodine receptor: a pore-
forming segment? Proc Natl Acad Sci U S A 96: 3345-3347.
Bauerova-Hlinkova V, Hostinova E, Gasperik J, Beck K, Borko L, Lai FA, Zahradnikova A,
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0
Identifying Enzyme Knockout Strategies on
Multiple Enzyme Associations
Bin Song
1
, I. Esra Byktahtakn
2
, Nirmalya Bandyopadhyay
1
,
Sanjay Ranka
1
and Tamer Kahveci
1
1
CISE Department, University of Florida, Gainesville
2
Systems and Industrial Engineering, University of Arizona, Tucson
USA
1. Introduction
Many biochemical engineering applications in drug discovery, food generation and cosmetic
production, aim to modify the metabolism of a given organism to increase or decrease the
production of a specic compound or a set of compounds. For example:
1. Fatty acid biosynthesis pathway converts fatty acids that are used in the cosmetic industry
in creams and lotions.
2. Butanoate metabolism produces poly--hydroxybutyrate which is essential for producing
plastics.
3. Mevalonic acid pathway and MEP/DOXP pathway produce carotenoid that are often used
as anti-oxidant in food industry. The metabolisms of many organisms, such as bacteria,
algae and plants naturally produce these compounds. A common practice is to extract
them from these organisms.
Enzymes play a signicant role in metabolism. They catalyze the chemical reactions that
transform a set of substrates (i.e., input compounds) into products (i.e., output compounds).
Metabolic engineering techniques often aim to manipulate a small set of genes to alter the
speed of the targeted enzymatic reactions. Their eventual goal is to reach a desired level
of compound concentrations produced or consumed by these reactions. One way to alter
the speed of the reactions dramatically is to knockout a set of enzymes. When an enzyme
is knocked out, it cannot catalyze a subset of the reactions, resulting in changes to the
productions of compounds.
When detailed in silico models are available, computational methods can be successfully used
to determine the enzyme set to knockout. These methods, when applicable, have much
lower time and cost requirements as compared to in vitro or in vivo experiments conducted
in wet labs. Wet-lab experiments often require substantial effort and time of the domain
experts and overall time requirements may be hours to several days. Moreover, the cost of
wet-lab experimentation signicantly increases when the number of enzymes that needs to
be knocked out is more than one. Manipulations that involve four to six enzymes are not
uncommon. As a result, biologists often employ computational methods as a preprocessing
step to lter out less promising compounds.
17
A number of heuristic in silico solutions exist to nd a promising set of enzymes. However,
nding the set of enzymes whose knockout leads to achieving the optimal compound
production rate is a computationally difcult problem. The number of possible subsets of
enzymes that can be considered for manipulation grows exponentially with the number of
enzymes in the pathway. Even if the size of each potential subset is limited to at most four, the
number of possible subsets for a pathway consisting of 500 enzymes is more than 2.5 billion.
Therefore, efcient methods that avoid inspecting the entire search space are necessary.
In order to nd a promising set of enzymes to knock out, we rst need to provide a
computational method to evaluate the metabolic system after some enzymes are knocked
out. There are several models to simulate the steady state of a metabolic network. We
categorize these methods into three different groups named, boolean models, linear models
and non-linear models. Boolean models can be an oversimplication of the metabolic
network, especially if the number of reactions and their connectivity increase. Non-linear
models require additional information about the network, which may not be available. Flux
Balance Analysis, (FBA) is a popular linear model which is widely used to compute the ux
distribution on the steady state of metabolic networks (Bonarius et al., 1997; Forster et al.,
2003; Kauffman et al., 2003). Segre et al. presented a quadratic programming method, named
minimization of metabolic adjustment(MOMA) (Segre et al., 2002). Shlomi et al. described
a MIP method, called regulatory on/off minimization (ROOM) for predicting the metabolic
steady states after the gene or enzyme knockouts (Shlomi et al., 2005).
It is easy to use these models to determine the impact on the metabolism, when a given set of
genes are knocked out. However, as discussed earlier, we are interested in nding the subset
of enzymes that lead to a desired impact. Optknock (Burgard et al., 2003), OptReg (Pharkya
& Maranas, 2006) and OptStrain (Pharkya et al., 2004) are three MIP based methods for
identifying the enzymes to be knocked out for the FBA model. All these methods make
the simplifying assumption that each reaction can be catalyzed by only one enzyme. This
simplication allows a quick conversion of the underlying variables using linear constraints,
where MILP or quadratic programming can be used to solve the problem. However, in
real metabolic networks, more than one enzyme can be involved in catalyzing a reaction.
In particular, more than two enzymes can substitute each other or work collaboratively to
catalyze a reaction. Figure 1 illustrates this on a real example we adopted from Reed et
al. (Reed et al., 2003). Here we describe these two kinds of enzyme collaborations in brief.
Collaborative enzymes: Some reactions require the presence of two or more proteins
or enzymes simultaneously. We call such enzymes as collaborative enzymes. In this case,
absence of even one of these enzymes is sufcient to slow down or stop the reaction.
Logically, there is an Boolean AND relation among these enzymes. In Figure 1 (top
portion), D-Xylose ABC Transporter is responsible for exporting/importing a variety of
molecules to and from bacteria. To carry out this function the genes XylF, XylG and XylH
jointly work to catalyze the reaction XYLabc.
Substitute enzymes: Two or more enzymes can substitute each other in catalyzing a
reaction. We call such enzymes as substitute enzymes. In this case, the presence of one of
the substitute enzymes sufces to carry out that reaction. Logically, there is a Boolean OR
relation among these enzymes. In Figure 1 (bottom portion), Glyceraldehyde 3-Phosphate
Dehydrogenase works in a number of metabolic pathways such as the Glycolysis /
Gluconeogenesis pathway or biosynthesis of phenylpropanoids. In a number of organisms
such as Arabidopsis thaliana (A. thaliana) this can be done by GapA or GapC with OR
association.
Identifying Enzyme Knockout Strategies on Multiple Enzyme Associations 3
One can easily generalize the notion of collaborative and substitute enzymes. Thus, a complex
topology consisting of multiple enzymes connected by a combination of OR and AND may
catalyze a reaction.
Protein
Reaction
Reaction
Protein
D-Xylose ABC Transporter
Glyceraldehyde 3-
Phosphate Dehydrogenase
XylF XylH XylG
&
XYLabc
GapA GapC
GAPD
Fig. 1. The gure depicts two examples of reactions catalyzed by multiple enzymes. In the
top portion, D-Xylose ABC Transporter is responsible for exporting/importing a variety of
molecules to and from bacteria. To carry out this function the genes XylF, XylG and XylH
jointly work to catalyze the reaction XYLabc with AND association. In the other portion,
Glyceraldehyde 3-Phosphate Dehydrogenase works in a number of metabolic pathways such
as the Glycolysis / Gluconeogenesis pathway or biosynthesis of phenylpropanoids.
Our goal aims to nd the optimal set of enzymes in the presence of multiple enzymes jointly
catalyzing the same reaction to knock out so that the production of the system is optimal. In
summary, the main contributions of this chapter are as follows:
We prove that the problem of nding the optimal enzyme set to knockout using
MIPL-based approaches is NP-hard even when only one enzyme catalyzes each reaction.
This proof is also corroborated by the fact that when the network size increases, the
execution time of Optknock framework increases exponentially.
We develop two solutions to deal with multiple enzyme association along with linear
constraints. Our solutions eliminate the limitation that each reaction is catalyzed by a
single enzyme. In our model, we allowmultiple substitute and collaborative enzymes. Our
rst solution uses a small number of binary variables in the underlying MILP formulation.
The second method increases the number of binary variables but requires a smaller number
of constraints. Inclusion of multiple enzymes signicantly extends the applicability of our
methods, as in real networks, multiple enzymes can catalyze a reaction.
Our experiments using the synthetic and real datasets demonstrate that allowing multiple
enzymes to catalyze a reaction increases the computational cost of the solution as compared
355 Identifying Enzyme Knockout Strategies on Multiple Enzyme Associations
to the cases when all reactions are catalyzed by a single enzyme. In our experiments, we
observe that our second method that introduces extra binary variables is signicantly superior
to our rst method in terms of execution time. These results also demonstrate that the enzyme
topology can have a substantial inuence on the performance of the MILP solution.
The rest of the chapter is organized as follows. Section 2 discusses the related work for this
chapter. Section 3 proves that nding the optimal set of enzymes to knock out using MILP is
NP-hard even when we allow only one enzyme to catalyze each reaction. Section 4 describes
the proposed methods when a reaction is catalyzed by multiple enzymes. Section 5 discusses
experimental results. We conclude our discussion in Section 6.
2. Related work
In order to identify a promising set of enzymes to knock out, we rst require a computational
method to evaluate the state of the metabolic system after multiple knockouts. There are
several models to simulate the steady state of a metabolic network. These methods can be
classied into three categories named Boolean models, linear models and non-linear models.
- Boolean Models: Boolean models consider each enzyme as a boolean variable. Each
variable can take a either true or false value representing whether the corresponding
enzyme is active or inactive. Each reaction is a boolean predicate that depends on
these variables. A reaction takes place only if its predicate evaluates to true. Sridhar
et al. and Song et al. propose a boolean model of the enzyme knockout strategy (Song
et al., 2007; Sridhar et al., 2007; 2008). These methods require the user to supply a list
of targeted compounds along with a metabolic network. The goal is to identify the set
of enzymes whose deletion stop producing all the targeted compounds while causing
minimumdamage. Here, we dene damage as the number of non-targeted compounds that
are eliminated because of the knockouts. Minimum damage is dened as the minimum
number of non-targeted compounds eliminated from the metabolism while eliminating
the targeted compounds given all possible ways of eliminating the targeted compounds.
Sridhar et al. propounds an optimal algorithm for this model (Sridhar et al., 2008). Song et
al. discusses a heuristic algorithm for nding the knockout enzyme strategy (Song et al.,
2007). Klamt et al. nds the enzymes for knockout by nding a minimal set of reactions
whose deletion leads to an infeasible balanced ux distribution. It employs a minimum
cut approach to solve the problem (Klamt & Gilles, 2004).
- Linear models: Boolean models can be an oversimplication of the metabolic network,
specially when the number of reactions and their connectivity increase. Flux Balance
Analysis, (FBA) is a popular technique used to analyze the steady state of metabolic
networks (Bonarius et al., 1997; Forster et al., 2003; Kauffman et al., 2003). FBA describes
a metabolic network as a set of linear equations. FBA nds an optimal steady-state
ux distribution that maximizes growth rate under constraints such as mass balance and
capacity. FBA achieves a successful description of the metabolic state system by predicting
growth rate and by-products of the metabolism (Edwards & Palsson, 2000a;b; Kauffman
et al., 2003). However, FBA may not be able to predict an accurate metabolic state after
gene or enzyme knockouts. Segre et al. presents a quadratic programming method named
minimization of metabolic adjustment (MOMA) for simulation of the resultant state after
knockouts (Segre et al., 2002). MOMA attempts to minimize the changes between the
ux distribution after a knockout. MOMA uses linear constraints such as mass balance,
capacity, and knockout constraints, which are the same set of constraints used by FBA.
Shlomi et al. describes a mixed integer programming method, named regulatory on/off
minimization (ROOM), for predicting the metabolic steady states after gene or enzyme
knockouts (Shlomi et al., 2005). ROOM nds the ux distribution which minimizes the
number of signicant ux changes from the wild-type ux distribution. Experiments
demonstrate that MOMA and ROOM are superior to FBA in their ability to predict the
resultant states after gene or enzyme knockouts. Optknock is an enzyme knockout strategy
based on the FBA model (Burgard et al., 2003). It uses a bi-level programming framework
for identifying the enzymes to be knocked out. In the inner level, the optimization nds
the ux distribution for a given cellular objective such as maximization of biomass yield
or minimization of metabolic adjustment (MOMA) (Alper et al., 2005; Segre et al., 2002)
etc). In the outer level, the optimization nds the enzymes to be knocked out to optimize a
biological objective (e.g., chemical production). OptReg is another bilevel programming
method for the enzyme knockout strategy (Pharkya & Maranas, 2006). The difference
between Optknock and OptReg is that Optknock framework considers only two states
(knockout vs non-knockout) for each reaction which are controlled by enzymes. However,
OptReg considers three sets of binary variables for each reaction. These correspond to
knockout or non-knockout and down regulation or up regulation. Thus, OptReg provides
more candidate manipulation solutions for enzymes. OptStrain, a MILP based method,
identies desired phenotypes by adding or deleting genes or enzymes (Pharkya et al.,
2004). All of the above methods use a MILP or a quadratic programming method.
Although the objective function of these methods may not be linear, the constraints are
linear.
- Non-linear models: Although less prevalent, these methods are also used to describe
metabolic networks. These methods incorporate further details about the network and
thus can simulate the cell systembetter than the linear model. S-systems (Savageau &Voit,
1987; Voit, 2000) and GMA model (Peschel & Mende, 1986; Voit, 2000) are two examples
of non-linear models for metabolic networks. Song et al. proposes methods for these
non-linear models (Song et al., 2011). Patil et al. presents an evolutionary programming
method which can be applied to non-linear models (Patil et al., 2005). These heuristic
solutions use non-linear models with non-linear constraints. They are not guaranteed
to produce optimal solutions. Also, the non-linear constraint models require additional
information about the network, which may not be available. Therefore, in this chapter we
still focus on the linear constraint model.
The methods described in this chapter use a linear model. Our major contribution, as
discussed already, is to allow multiple enzymes to catalyze a reaction. This signicantly
extends the usability of such methods, as in real networks more than one enzymes can catalyze
a reaction.
3. Problem formulation
Given a metabolic network and an objective function, one standard way to nd the optimal
set of enzyme knockouts is to solve the problem as an MILP which is modeled using FBA. In
this section, we focus on the MILP formulation of the enzyme knockout problem and prove
that the problem is NP-hard even when a single enzyme catalyzes each reaction.
3.1 Formulation
Given a set N = {1, ...,

N} of N metabolites and a set M = {1, ...,

M} of M metabolic
reactions, our goal is to determine the maximum yield of the desired products in a metabolic
network while minimizing the enzyme knockout costs. We summarize the decision variables
as follows:
v
j
: the ux of reaction j;
y
j
: binary variable which equals to 0 if an enzyme in reaction j is knocked out,
and 1 otherwise.
Other relevant parameters used in this problem are:
S
ij
: stoichiometric matrix coefcient of metabolite i in reaction j;
l
j
: minimum possible ow corresponding to ux j;
u
j
: maximum possible ow corresponding to ux j;
h
j
: cost of blocking the enzyme corresponding to reaction j;
w
j
: weight corresponding to the value of ux j.
Here, l
j
and u
j
are estimated by minimizing and maximizing every reaction ux subject to the
constraints from the enzyme knockout ux balance model (EKFB) framework given below.
Let I be a set of external metabolites that are imposed on the pathway, and J be the set of
metabolites that will not be used within the pathway once they are produced. We denote
the ux of the source metabolites in the metabolic pathway by b
i
and the ux of the sink
metabolites by c
i
.
Given these variables and parameters, we represent the integer programming formulation for
EKFB as follows:
max

jM
w
j
v
j

jM
h
j
(1 y
j
) (1)
s.t.

jM
S
ij
v
j
=
b
i
if i I;
c
i
if i J;
0 if i N\ {J I}.
(2)
l
j
y
j
v
j
u
j
y
j
j M (3)
jM
(1 y
j
) K, j M (4)
y
j
{0, 1} j M. (5)
The objective function (1) maximizes weighted ux less xed charge corresponding to
the enzyme knockouts. Constraint (2) provides ux balance equations dened by the
stoichiometric matrix. Constraint (3) includes the xed charge variable y
j
. If the enzyme
corresponding to reaction j is knocked out, the value of the ux is set to zero and a xed
charge h
j
for knocking out the enzyme is imposed. If the xed charge variable y
j
takes value
1, then the lowest ux value is l
j
while the highest possible ux value is u
j
. Constraint (4)
imposes the condition that the maximum number of knockouts is K. Constraints (5) enforce
integrality on the xed charge variables. Similar formulations are provided in Burgard et
al. (Burgard et al., 2003), Cover et al. (Covert et al., 2001) and Palsson (Palsson, 2000).
3.2 NP completeness
To prove that nding the enzymes to knockout by EKFB is NP-hard, we show that the
uncapacitated xed charge network ow problem, which is NP-hard, is a special case of the
EKFB (Ng & Rardin, 1996). Let G = (V, A) be a directed graph, where V is the set of nodes,
A is the set of arcs, s V is the single source node, T V is a collection of sink vertices and
d
t
> 0 is the demand for node t. Let x
ij
denote the ow on arc (i, j) with a cost c
ij
. Let the
variable z
ij
be equal to 1 if arc (i, j) is selected with a xed cost f
ij
and 0 otherwise. We then
dene the uncapacitated xed charge network ow problem (UFNF) as the problem of nding a
set of arcs that allow a supply node to send resources to a set of demand nodes, such that the
sum of xed and variable costs are minimized. UFNF can be formulated using the following
mixed-integer program:
min

(i,j)A
f
ij
z
ij
+

(i,j)A
c
ij
x
ij
(6)
s.t.

(i,k)A
x
ik

(k,j)A
x
kj
=
tT
d
t
if k = s;
d
k
if k T;
0 if k V\ {T s}.
(7)
x
ij
z
ij
(i, j) A (8)
x
ij
0 (i, j) A (9)
z
ij
{0, 1} (i, j) A. (10)
The objective function (6) minimizes the sum of the xed costs associated with selecting arc
(i, j) and variable costs for sending ow through (i, j). Constraints (7) are classical ow
conservation constraints. Constraints (8) ensure that there can not be any ow if z
ij
is 0. Also,
the maximum ow can be at most if z
ij
is 1. Constraints (9) and (10) ensure that x
ij
is
nonnegative and z
ij
is binary respectively.
Theorem 1. Finding the enzyme knockout strategy by EKFB is NP-Hard.
Proof: Let N
be the set of the metabolites and M
be the set of the reactions in a special case

metabolic pathway in EKFB. We model it as a network graph G
= (N
, M
), where each node

represents a metabolite i N
and each arc represents a reaction k M
using metabolite i to
produce metabolite j.
For i N
and k M
, we redene the stoichiometric matrix S

ik
as S
ij
(i, j N
) such that
(i, j) represents reaction k as follows:
S
ij
=
1 if (i, j) M
;
1 if (j, i) M
;
0 otherwise.
(11)
Note that S
ij
has entries 1, 1, and 0, and thus is a special case of S
ik
. We also dene a new
variable v
ij
as the ux corresponding to reaction k M
. Let I
I be a set of external
metabolites that are imposed to the pathway, and J
J be the set of metabolites that will

not be used within the pathway after they are produced. Let us dene a parameter

b
i
such
that b
i
=

b
i
and c
i
=

b
i
for each i N
. By using the stoichiometric matrix S
ij
and the new
variables v
ij
, the constraint (2) can be written as,
(i,l)M
v
il

(l,j)M
v
l j
=
b
l
if l I
b
l
if l J
;
0 if l N
\ {I
}.
(12)
We now dene a binary variable z
ij
for each variable y
k
, which assumes value 1 if the arc (i, j)
is selected and 0 otherwise. We dene costs c
ij
and

f
ij
such that c
ij
= w
k
,

f
ij
= h
k
. Finally,
we dene a constant

as

= u
k
, and set l
k
= 0 for each reaction k M
, which is dened by
the arc (i, j). Then, the constraint (3) can be written as,
0 v
ij

z
ij
(i, j) M
(13)
with an objective function,
min

(i,j)M
c
ij
v
ij
+

(i,j)M
f
ij
z
ij
(14)
Thus, a special case of EKFB with an objective function (14) and constraints (11), (12), (13) and
z
ij
{0, 1} is a UFNF and hence EKFB is NP-Hard.
4. Methods for multiple enzymes
In this section, we develop a more general version of EKFB where we allow multiple enzymes
to catalyze a reaction. This extension improves the applicability of our methods as in real
networks more than one enzymes can catalyze a reaction. In particular, we focus on the
constraints (3) and model the possible interactions between enzymes regarding the reactions
they catalyze.
Let E
i
be a Boolean variable that denotes whether the ith enzyme is active (i.e., E
i
= true) or
inhibited (i.e., E
i
= false). As discussed earlier, in EKFB, we assume that a reaction can be
catalyzed only by a single enzyme. We use the Boolean variable y
i
which is equal to 1 if an
enzyme is active, and 0 otherwise.
Let us denote the set of variables for the enzymes that are involved in catalyzing the ith
reaction with E
i
{E
1
, E
2
, , E
M
}. For simplicity, we will use the notation E
i
= {E
ij
| E
ij
{E
1
,
E
2
, , E
M
}} to denote this set. Let F
i
be a function on {0, 1}
|E
i
|
representing the relationship
between the enzymes for the ith reaction. This function takes E
i
as input and produces an
integer. It evaluates to 1 if the ith reaction takes place according to the values of the variables
in E
i
. It evaluates to 0 otherwise. Also, let the constants l
i
and u
i
represent the minimum and
the maximum ux values. We write the second set of constraints as:
l
i
F
i
v
i
u
i
F
i
. (15)
Depending on association between the enzymes that catalyze a reaction, we formulate F
i
for
three different scenarios.
A topology consisting only of substitute enzymes that catalyze any reaction. Each reaction
may be catalyzed by a single enzyme or a set of enzyme based on the OR association i.e.,
only one of the enzymes need be present to catalyze the reaction (Section 4.1).
A topology consisting only of collaborative enzymes that catalyze any reaction. Each
reaction may be catalyzed by a single enzyme or a set of enzymes based on the AND
association i.e., all of the enzymes need to be present to catalyze the reaction (Section 4.2).
A complex topology consisting of multiple enzymes related by a combination of OR and
AND may catalyze a reaction (Section 4.3).
Shlomi et al. presents a way of replacing Boolean expressions that contains two Boolean
variables with linear inequalities (Shlomi et al., 2007). However, as the number of Boolean
variables grows, the number of additional variables required by this method grows rapidly
making the problemnontrivial. In the following sections we discuss two alternative strategies
to deal with each of these three scenarios. We name these strategies the Binary Method and
Continuous Method. The former one introduces additional Boolean variables. The second one
avoids the addition of Boolean variables, but comes at the expense of additional constraints.
We discuss these in detail in the following sections.
4.1 MILP solution in the presence of substitute enzymes
In this section, we consider the case when all the enzymes that catalyze the same reaction can
substitute each other. In this case, the presence of at least one of the substitute enzymes is
sufcient to carry out the corresponding reaction. Let E
i
= {E
ij
| E
ij
{E
1
, E
2
, , E
M
}} denote
a set of variables representing the substitute enzymes for reaction i (i.e., ux v
i
). Then we
write the function F
i
that governs the relationship between the variables in E
i
as:
F
i
= max
E
ij
E
i
{E
ij
}
Thus the constraint (15) becomes:
l
i
max
E
ij
E
i
{E
ij
} v
i
u
i
max
E
ij
E
i
{E
ij
}. (16)
We address the problem of nonlinearity in constraint (16) by performing a variable
transformation, which leads to a set of linear constraints. We solve them using traditional
MILP solution techniques such as simplex method.
Our linearization technique considers lower and upper bounds separately. We linearize lower
bounding constraints given by the inequality l
i
max
E
ij
E
i
{E
ij
} v
i
as follows,
l
i
E
ij
v
i
E
ij
E
i
. (17)
Linearization of the upper bounding constraints given by the inequality v
i
u
i
max
E
ij
E
i
{E
ij
}
is more complex compared to that of the lower bound. For the linearization, we consider two
approaches, namely binary and continuous methods.
Binary method: In this method, we propose the following linear constraints in order to
enforce binary restrictions on F
i
(i.e., F
i
{0, 1}):
F
i

j
E
ij
n
i (18a)
F
i

j
E
ij
i (18b)
F
i
{0, 1} i (18c)
Continuous method: In this method, we dene F
i
using a continuous variable that takes
value in the real domain. We replace the upper bound constraint v
i
u
i
max
E
ij
E
i
{E
ij
} with
the following linear constraints:
F
i

j
E
ij
i (19a)
F
i
1 i (19b)
F
i
E
ij
i, j (19c)
The constraints (19b)- (19c) enforces F
i
to assume a binary value, even though we do not
directly impose binary restrictions on it.
4.2 MILP solution in the presence of collaborative enzymes
In this section, we consider the case where multiple enzymes collaborate with each other to
catalyze the same reaction. In this case, all the enzymes are necessary for the reaction to
initiate. Let E
i
= {E
ij
| E
ij
{E
1
, E
2
, , E
M
}} denote a set of variables representing the
substitute enzymes for reaction i (i.e., ux v
i
). We write the function F
i
that governs the
relationship between the variables in E
i
as:
F
i
= min
E
ij
E
i
{E
ij
}
Thus, we write constraint (15) as,
l
i
min
E
ij
E
i
{E
ij
} v
i
u
i
min
E
ij
E
i
{E
ij
}. (20)
As we discussed in Section 4.1, constraint (20) is nonlinear. We linearize this constraint using
additional variables. We address lower and upper bounds separately.
First, we focus on the upper bound constraints given by the inequality v
i
u
i
max
E
ij
E
i
{E
ij
}.
We linearize this part without introducing new variables as follows:
v
i
u
i
E
ij
E
ij
E
i
(21)
The linearization of the lower bound constraints given by the inequality l
i
min
E
ij
E
i
{E
ij
} v
i
is more complicated. Analogous to the substitute enzyme case, we develop both binary and
continuous methods presented in the following two sections.
Binary method: We have already assumed that F
i
{0, 1}. We linearize the nonlinear
constraint l
i
min
E
ij
E
i
{E
ij
} v
i
under this assumption as follows:
F
i

j
E
ij
n
i (22a)
F
i
>
j
E
ij
n
1 i (22b)
F
i
{0, 1} i (22c)
Continuous method: For the continuous method, we replace the lower bound constraint
l
i
min
E
ij
E
i
{E
ij
} v
i
with the following linear constraints:
F
i
E
ij
i (23a)
F
i

j
E
ij
(n 1) i (23b)
F
i
0 i (23c)
4.3 MILP solution in the presence of complex association of enzymes
In this subsection, we generalize the methods described in the previous two subsections in
order to allow associations with arbitrary forms. We consider the case when the reaction can
be catalyzed by a set of enzymes such that some of them can substitute for each other and
others need to work collaboratively.
For example, assume that ith reaction can be catalyzed by two alternative enzyme complexes
that can substitute each other. Also assume that the rst and the second of these complexes
are formed from two and three enzymes, respectively. These two or three enzymes in the
complexes collaborate with each other. We formulate this relationship as F
i
= max { min {E
i1
,
E
i2
}, min {E
i3
, E
i4
, E
i4
} }.
Using standard rules from Boolean algebra, all Boolean equations can be written into
disjunctive or conjunctive normal forms. Thus, we transform the equation for each reaction
into the following form:
F
i
= max
E
k
i
{ min
E
ij
E
k
i
{E
ij
}}. (24)
In this equation, E
k
i
denotes the kth set of collaborative enzymes required by the ith reaction.
Thus, we have
k
E
k
i
= E
i
. We dene a new binary variable Z
k
i
{0, 1} corresponding to each
E
k
i
and rewrite Equation (24) as,
F
i
= max
E
k
i
Z
k
i
. (25)
where,
Z
k
i
= min
E
ij
E
k
i
{E
ij
}. (26)
The methods in Section 4.1 and Section 4.2 are used for constraints (26) and (25) respectively
to linearize the constraint (15).
5. Experiments
In this section, we evaluate the performance and the limitations of our methods on real and
articially generated metabolic networks. The synthetic datasets provide us a controlled
simulation environment that allows us to determine the impact of different characteristics
of the network on the performance of our algorithms. We evaluate the performance of our
methods quantitatively in terms of their execution time (in seconds).
5.1 Datasets
In our experiments, we used the following real and synthetic datasets.
- Synthetic datasets: We randomly generated ten networks of different sizes (given by the
number of compounds and the number of reactions). In order to simulate the real
networks accurately, we generated these networks so that the number of reactions that
involve a compound is distributed according to the power law distribution (Voit, 2000). In
other words, the probability of the number of reactions that each compound involves in
decreases exponentially with the number of reactions.
In order to evaluate the impact of multiple enzymes for catalyzing a reaction, on the
performance of the algorithms, we generated two types of datasets:
Single enzyme dataset: In this dataset, each reaction is catalyzed by only one enzyme.
Thus, the number of enzymes is equal to the number of reactions.
Multiple enzyme dataset: In this dataset, all the reactions are catalyzed by at least one
enzyme. The number of enzymes attached to a reaction is based on the power law
distribution: the probability that a reaction is catalyzed by k enzymes decreases exponentially
with k. Roughly, 40% of the reactions are catalyzed by at least two enzymes; 30% of the
reactions are catalyzed by at least three enzymes; 23.5% of reactions are catalyzed by at
least four enzymes; 18.5% of reactions are catalyzed by at least ve enzymes and 5% of
reactions are catalyzed by at least nine enzymes. Based on these probabilities, we build
ten synthetic networks for each network size. Section 5.2.1 describes the results for the
synthetic datasets.
- Real dataset: We use the metabolic pathways of Homo sapiens (H. sapiens) from
KEGG (Kanehisa & Goto, 2000). The entire H. sapiens metabolism consists of 640 enzymes,
1176 reactions and 1067 compounds. Section 5.2.2 provides the results for these real
datasets.
Experiment platform: We implemented our algorithms in C++. We applied ILOG CPLEX
11.2 to nd the integer linear programming solutions. We executed our experiments on a
system with two Pentium 4 3.2Ghz and 1M cache processors, 6 gigabytes of RAM, and a
Linux operating system.
5.2 Results
In this section, we evaluate the performance of our algorithms on the synthetic (Section 5.2.1)
and real datasets (Section 5.2.2).
5.2.1 Evaluation on synthetic datasets
Our goal in this section is to evaluate the performance of our algorithm for a variety
of network parameters using synthetic datasets. These experiments can be decomposed
into two sets as described in the previous subsection, namely, single enzyme dataset and
multiple enzyme dataset. For an effective comparison, we use identical topology of reactions
and compounds for both multiple and single enzyme set. We consider two cases for the
multiple enzyme set: a) All multiple enzymes substitute each other. b) All multiple enzymes
collaborate with each other.
Performance analysis on single enzyme set: Section 3 proves that nding the enzyme
knockout strategy using MILP is NP-Hard. Consider the case when only one enzyme
0.01
0.1
1
10
100
1000
10000
100000
50 100 150 200 300
#R
E
x
e
c
u
t
i
o
n

t
i
m
e

(
s
e
c
)
#C = #R*25%
#C = #R* 50%
Fig. 2. The average execution time (in seconds) for the networks on single enzyme set. #R
denotes the number of reactions and #C denotes the number of compounds in the network.
The execution time grows exponentially as the number of reactions increases for both the
cases and can be prohibitive even for a few hundred reactions.
catalyzes a reaction. We conduct our experiments using the MILP formulation for two
different settings. In the rst setting, the number of compounds is 25% of that of the reactions,
while for the second setting, it is 50%. Figure 2 plots the average execution times for networks
with different number of reactions.
The execution time grows exponentially as the number of reactions increases for both the cases
and can be prohibitive even for a few hundred reactions. This time constraint necessitates
the advent of heuristic methods for large networks. Also, we observe a steep increase in
execution time for larger number of compounds. For the same number of reactions, doubling
the number of compounds leads to an overall time increase by several orders of magnitude. It
can be concluded that, heuristic methods which can reduce the number of compounds from
the constraint set, can have the potential to improve the execution time of the MILP solutions.
Performance analysis for multiple enzymes set: The results in the previous section (along
with the NP-hardness of the problem) show that the MILP solution has exponential execution
time complexity in terms of the network size. We now study performance of our two
solutions with multiple enzymes per reaction. In this experiment, we study the running time
requirements in the presence of multiple substitute and collaborative enzymes. We compare
these times to those of single enzymes. Note that, the comparison against single enzyme
favors the single enzyme dataset as it has fewer variables. This, however, should serve as a
lower bound for execution time for the multiple enzyme cases. We summarize the result as
follows:
1. Binary method: Figure 3 depicts the results of our binary method for variable number of
compounds and reactions. The results demonstrate that the presence of multiple enzymes
0.01
0.1
1
10
100
(12, 50) (25, 50) (50, 100) (25, 100) (50, 200)
Test cases (#C, #R)
E
x
e
c
u
t
i
o
n

t
i
m
e

(
s
e
c
)
single enzyme set
multiple enzyme set - substitute
multiple enzyme set - collaborate
Fig. 3. The average execution time (in seconds) for the networks with single and multiple
enzymes. All multiple enzymes cases are either all substitutions or all collaborations. For
multiple enzymes set, we use binary method. The results demonstrate that the presence of
multiple enzymes increases the execution time signicantly as compared to the case when
only a single enzyme catalyzes a reaction.
increases the execution time signicantly as compared to the case when only single enzyme
catalyzes a reaction. This improvement holds true for both substitute and collaborative
enzymes. The running time for multiple enzymes is two to 16 times that of the single
enzyme case. In most of the test cases, collaborative enzymes resulted in a higher increase
in execution time.
2. Continuous method: Figure 4 shows the execution time of multiple enzymes set by
continuous method and that for the single enzyme set. Similar to the binary method,
multiple enzymes set requires much more time than that of the single enzyme set. As
the network size increases, the gap between the execution time of the multiple enzymes
set and that of the single enzyme set increases exponentially. This suggests that the
presence of multiple enzymes necessitates heuristics solutions for large networks. Also,
collaboration among enzymes requires relatively higher execution time as compared to
that of the substitution between enzymes in majority of the experiments.
3. Comparison of the two methods: Recall that the binary method introduces additional
binary variables to linearize the constraints. The continuous method only generates
additional continuous variables. However, it requires additional constraints. Our
experiments (see Figures 3 and 4) demonstrate that the Binary method executes twice or
more faster than the continuous method for the case when all multiple enzymes cases are
substitutions. When the multiple enzymes collaborate with each other, the gap between
0.01
0.1
1
10
100
1000
10000
(12, 50) (25, 50) (50, 100) (25, 100) (50, 200)
Test cases (#C, #R)
E
x
e
c
u
t
i
o
n

t
i
m
e

(
s
e
c
)
single enzyme set
multiple enzyme set - substitute
multiple enzyme set - collaborate
Fig. 4. The average execution time (in seconds) for the networks with single and multiple
enzymes. All multiple enzymes cases are either all substitutions or all collaborations. For
multiple enzymes set, we use continuous method. As the network size increases, the gap
between the execution times of the multiple enzymes set and the single enzyme set increases
exponentially.
the running time of the binary and continuous method increases further. Therefore, for the
large networks, binary method is the preferred choice.
5.2.2 Evaluation on the real dataset
In this section, we evaluate the performance of our algorithm on real metabolic networks
taken from the KEGG database. We use the metabolisms of H. sapiens. Given, the superior
performance of the binary method over continuous method (as described in the previous
subsection), we limit ourselves to the binary method on the real dataset. We execute the binary
method for purine metabolism, metabolismof cofactors and vitamins, amino acid metabolism
and the entire metabolism. However, the KEGG database does not provide the details of
enzyme association information. Thus, we consider two alternative cases: a) all the enzymes
are collaborations, b) all the enzymes are substitutions. Table 1 demonstrates the running time
using the binary method. These results show that our method requires less than one second
of execution time and hence, are scalable to practical network sizes for both cases. Even for
the entire metabolism of H. sapiens, the execution time is less than half a second. This makes
our methods of great practical importance.
It is worth mentioning that the execution times on the real datasets are substantially lower
than that of the synthetic datasets. This is because, the topology of the real networks is much
sparser than the ones we used for our synthetic experiments. Therefore, less time is required
to nd the ux distribution on the real networks.
Pathway #E #R #C Collaborative Substitute
Purine metabolism 52 92 65 0.07 0.13
Metabolism of cofactors and vitamins 90 132 122 0.04 0.05
Amino acid metabolism 195 317 305 0.05 0.06
the entire metabolism 640 1176 1067 0.38 0.28
Table 1. Execution time in seconds of our binary method for the metabolisms of H. sapiens
from KEGG. #E, #R and #C denote the number of enzymes, reactions and compounds
respectively in the metabolism. The results demonstrate that our method requires less than
one second of execution time. Hence it is scalable to practical network sizes for both the
cases.
6. Conclusions
Given a metabolic network and a goal, such as maximizing or minimizing the production of
a set of compounds, we considered the problem of computationally determining the optimal
enzyme knockouts to modify the production of compounds using the Flux Balance Analysis
(FBA) model. We proved that the problem of nding the optimal enzyme set to knockout is
NP-hard even when only one enzyme catalyzes a reaction.
We developed two strategies to identify the enzymes to knockout, when multiple enzymes
catalyze a single reaction. We allowed multiple substitute and collaborative enzymes. In the
proposed solutions, we eliminate this limitation of single enzyme. Our rst solution uses a
small number of binary variables in the underlying MILP formulation. The second method
increases the number of binary variables but requires a smaller number of constraints.
Our experiments using synthetic and real datasets demonstrated that adding extra binary
variables is signicantly superior to adding additional constraints in terms of execution time.
For the metabolism consisting of all the pathways of H. sapiens, our binary method requires
less than one second. This makes our methods of great practical importance.
We believe that the approach presented in this chapter is not limited to MILP based strategies.
It should also be applicable to other linear constraint strategies, e.g. quadratic programming,
where the objective function is non-linear but the constraints are linear.
7. Acknowledgment
This work was supported partially by NSF under grants CCF-0829867 and IIS-0845439.
8. References
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Part 6
Genome Analysis

18
Using Bacterial Artificial Chromosomes to
Refine Genome Assemblies and to Build
Virtual Genomes
Abhirami Ratnakumar
1,2
, Wesley Barris
1,3
,

Sean McWilliam
1
and Brian P. Dalrymple
1

1
CSIRO Livestock Industries, St. Lucia, QLD
2
now at the Department of Medical Biochemistry and Microbiology,
Uppsala University, Uppsala
3
now at Cobb-Vantress, Siloam Springs, Arkansas
1
Australia
2
Sweden
3
USA
1. Introduction
Recent years have seen an explosion in the sequencing of genomes, including those of
ruminants. A number of assemblies of the sequence of the bovine genome are now available
(Elsik, et al., 2009; Zimin, et al., 2009). Although the sheep genome sequence is not such a
high priority, the International Sheep Genomics Consortium (ISGC_website) has a long term
strategy to develop a number of tools for the application of genomics in sheep research and
breeding (Archibald, et al., 2010). We have demonstrated recently how comparative
genomics and Bacterial Artificial Chromosome (BAC)-libraries can be used to construct
detailed virtual genomes as a framework for genome assemblies of related species
(Dalrymple, et al., 2007). As new and improved genome assemblies of the genomes
contributing to an initial virtual genome assembly are produced, the virtual genomes will
need to be regularly updated to incorporate the latest available information. In the original
analysis, three genomes (bovine, dog and human) with various levels of coverage and stages
of assembly were used (Dalrymple, et al., 2007). With the availability of increasing numbers
of assemblies, the benefit of using more than three genomes, or the most appropriate
evolutionary distances of the genomes, is not immediately clear. Here we describe the
construction of a modified version of the bovine Btau3.1 assembly using cattle and sheep
BACs and the use of this assembly in the construction of an updated virtual sheep genome,
combining information from the original sheep virtual genome (vsg 1.2) and the horse
(Wade, et al., 2009) and dog (Lindblad-Toh, et al., 2005) genomes. The impact of inclusion of
additional genome sequences is analysed. The approach described here for sheep is an
example of an approach which can be applied more broadly to genomes of any source, for
example for the fish species, tilapia (Soler, et al., 2010) and catfish (Liu, et al., 2009). Indeed,
the same principles also apply to the detection of differences between different individuals
of the same species.


374
2. Materials and methods
2.1 Data sources and sequence search parameters
All genome sequences, except Btau3.#x versions, were downloaded from the UCSC
comparative genomics website (UCSC; Fujita, et al., 2011). The full set of BAC-end sequences
(BESs) from the CHORI-243 sheep BAC library, deposited in GenBank with the following
accession numbers; CL632218-CL639051, CZ920079-CZ926973 and DU169919-DU532729
(Dalrymple, et al., 2007), were filtered to remove duplicate sequences and to identify the set
of high confidence BACs (Ratnakumar, et al., 2010a). The filtered set of sheep BAC-end
sequences were aligned to the lower case masked versions of the bovine genome assembly
(Btau3.1) and the revised bovine genome assembly (Btau3.5x) using MegaBLASTn with the
following optimised parameters: -r 1 -q -1 -X 40 -W 8, as previously described (Ratnakumar,
et al., 2010b). The filtered set of sheep BESs were aligned to the lower case masked versions of
the dog genome sequence assembly (canFam2), the horse genome sequence assembly
(equCab1) and to the human genome sequence assembly (hg17) using BLASTn with the
following parameters: -W 7 -r 17 -q -21 -G 29 -E 22 -X 240 -e 1 -f 280 -F m -U T and -z
3076781887 (human) and -z 2531657226 (dog), as previously described (Dalrymple, et al.,
2007). No cut offs were applied to the BLAST output except that for each sheep BAC-end
sequence only the best hit from each of the genomes was used for the next steps. If two BESs
hits to the same genome assembly with equal scores were obtained the hit on the same
chromosome as the best hit for the BES determined from the other end of the BAC was
retained. If more than two hits with equal scores were obtained the BES hit was discarded.
The BESs from the CHORI-240 cattle BAC library, GenBank accession numbers; BZ830806-
BZ891831, BZ896446-BZ956676, CC447354-CC447937, CC466118-CC470858, CC470880-
CC596504, CC761663-CC775995, CG917936-CG918393, CG976420-CG992944, CL603252-
CL610093, CW848133-CW848163, CZ012846-CZ027312 (Snelling, et al., 2007), were aligned
to the cattle and virtual sheep genome sequences using BLAT (Kent, 2002).
Bovine genome assembly Btau3.1 sequence contigs were aligned to the human, dog and
horse genomes using MegaBLASTn as described above.
2.2 Genome coordinate conversion
The coordinates from the mapping of the sheep BESs to the dog and human genomes were
converted to the framework of the bovine genome assembly Btau3.1 using the LiftOver
utility (LiftOver; Fujita, et al., 2011) and the canFam2 to Btau3.1 and hg17 to Btau3.1
coordinate conversion chain files respectively, also downloaded from UCSC genome
bioinformatics site (UCSC; Fujita, et al., 2011). If the initial application of LiftOver was not
successful for a region of the genome, regions of 100 bases either side of the BAC-end
sequence were taken and positioned using LiftOver (pseudoliftOver). If this was again
unsuccessful the process was repeated in steps of 100 bases until a successful application of
the LiftOver utility for a region was achieved, or a distance of 10kb was reached (Dalrymple,
et al., 2007).
Coordinate conversion (chain) files able to be read by the LiftOver utility to convert bovine
genome assembly Btau3.1 coordinates to bovine genome assembly Btau3.#x version
coordinates were built based on the revised order of Btau3.1 contigs and scaffolds in
Btau3.#x version. Similarly a coordinate conversion file to convert Btau3.5x coordinates to
virtual sheep genome assembly coordinates was built based on the order of Btau3.5x
scaffolds in the virtual sheep genome.
Using Bacterial Artificial Chromosomes
to Refine Genome Assemblies and to Build Virtual Genomes

375
2.3 Assigning BACs to groups and building BAC contigs
BACs were assigned to the groups; tail-to-tail, tail-to-head etc. on the basis of the
relative orientations of the two BESs from each BAC on the relevant genome assembly and
the distance apart of the BESs. Outsize BACs were those with the two BESs mapped to the
same chromosome in the relevant genome assembly and less than 10 kb, or more than 200
kb, apart. Data processing was undertaken using a series of Perl scripts. BACs with both
BESs mapped to the genome, but mapped to two different chromosomes, were assigned to
the breaks group. BACs with only one BES mapped to the genome were assigned to the
unpaired group.
BAC-comparative genomic contigs (BAC-CGCs) were constructed for the BACs from each
species mapped to each genome assembly using Perl scripts to process the data (Dalrymple,
et al., 2007). Starting from the beginning of each chromosome the first BAC that overlapped
with a second BAC was identified, the BAC-CGC was extended until no further overlapping
BACs were identified. This process was repeated along the chromosome until the last BAC
mapped on the chromosome was reached. The process was repeated for each chromosome
in the genome assembly.
2.4 Construction of Btau3.5x
Using Perl scripts and the data set of the mapping of the bovine BESs to the scaffolds of the
Btau3.1 genome assembly an initial minimization of the number of non-tail-to-tail BACs was
undertaken. The scripts started with the first scaffold on chromosome 1 of the assembly and by
testing the number of BAC links between this scaffold and all other scaffolds in the assembly
identified the most likely adjacent scaffold and the orientation of the scaffold based on
maximising the number of tail-to-tail BACs. Two or more linking tail-to-tail BACs without
overlapping BES mapping coordinates on both scaffolds were required to continue the chain.
Only high confidence bovine BACs (Ratnakumar, et al., 2009) were used in the assembly.
Adjacent scaffolds assigned to the same chromosome in the Btau3.1 assembly were preferred
over a more highly linked scaffold assigned to another chromosome, if the preferred scaffold
on the original chromosome was itself linked to an adjacent scaffold on the original
chromosome. If no scaffold assigned to the same chromosome as the rest of the chain was
linked into the chain by BACs, or the less strongly linked scaffold from the same chromosome
terminated the chain, the most highly linked scaffold from another chromosome was
incorporated. If the newly added scaffold was linked back to the original chromosome at the
next step of scaffold incorporation it was retained in the chain, otherwise the chain was
terminated and the chromosome changing scaffold was also removed from the scaffold chain.
For each scaffold in the chain BAC-links from both ends of the scaffold were assessed to
enable to inclusion of scaffolds preceding the initiating scaffold, or located between two
scaffolds in a chain, but which were only linked to an adjacent following scaffold. The
scaffold chain building process was continued until it was terminated with a scaffold not
linked by two or more BACs to another scaffold. The penultimate scaffold in the chain was
then tested for BAC links to a second scaffold and incorporated if it met the criteria
described above. The chain building process was then continued. If no second linked
scaffold could be identified the scaffold chain building was terminated. The next
unincorporated scaffold from the same chromosome of the Btau3.1 assembly was then used
to initiate the next scaffold chain. When all scaffolds from the first chromosome had been
tested the first scaffold from the next chromosome was used and the process repeated until
all scaffolds assigned to a chromosome of the Btau3.1 assembly had been tested.


376
Once the scaffold chain assembly had been completed the scaffolds not assigned to
chromosomes in the Btau3.1 assembly (the UnChr) were then linked into the scaffold chains
in a similar, but separate process. The resulting scaffold chains were then ordered and
oriented using the consensus of the mapping of the order of the BACs in the physical bovine
BAC map (Snelling, et al., 2007) to the BACs in the bovine scaffold chains. The initial data
set Btau3.1x was then displayed as a browseable genome using Gbrowse (Stein, et al., 2002)
to allow the integrity of the assembly of the scaffolds to be visually assessed. Genome
contigs, scaffolds, bovine BAC mapping positions were displayed as separate groups.
Clusters on non-congruent BACs (i.e. not tail-to-tail) identified regions with remaining
assembly problems.
Using Perl scripts and the data set of the mapping of the sheep BESs to the Btau3.1 genome
assembly, including BES mapping data integrated onto the Btau3.1 assembly from the horse,
dog and vsg1.2 assemblies sheep BACs were assigned to tail-to-tail etc. groups and
displayed on the Btau3.1x genome browser in a series of tracks. Positions of the BESs
mapped to the separate genomes were integrated on Btau3.1 as previously described
(Dalrymple, et al., 2007).
The mappings of the bovine genome assembly Btau3.1 sequence contigs to the human, dog
and horse genomes were displayed as separate tracks on the Btau3.1x genome browser using
the UCSC chromosome colour scheme (Fujita, et al., 2011) to identify the chromosome of best
match in the relevant species. Asymmetric symbols were used to represent the orientation of
the mapping of the contigs to the human, dog and horse genomes relative to the bovine
genome. The chromosomal coordinates of the mapping in the non-bovine genome were also
readily accessible to the users of the browser using mouse-over and mouse-click display boxes.
This information was used in the manual refinement of the assembly, in particular in the
definition of scaffold split points for the insertion of other scaffolds and/or the inversion of
small numbers of adjacent contigs within a scaffold, where extensive use was made of
comparative genomics information at the level of the sequence contigs.
Subsequently four major rounds of revision and refinement of the bovine genome assembly
were undertaken manually and decisions on the chromosomal assignment, order of
scaffolds and orientation of scaffolds and of sequence contigs were made based on the cattle
and sheep BAC mapping and the comparative genomics. Generally in cases of ambiguity
parsimony was applied. For the construction of each new version of the assembly changes
were recorded in an Excel spreadsheet and Perl scripts were used to convert the Excel
spreadsheet into a genome assembly agp file (AGP_file_specification). The agp file was used
to generate the sequence of the genome assembly, the coordinate conversion chain file (for
use by the LiftOver utility) and the contig and scaffold tracks for the genome browser
version for the new assembly. For each successive version of the revised assembly of the
bovine genome the manual revision was undertaken interactively using the tracks on the
genome browser to make decisions.
2.5 Construction of the virtual sheep genome vsg2.0
To generate the virtual sheep genome assembly the mid point between each pair of BAC-
CGCs built using sheep BACs on the bovine Btau3.5x genome assembly was identified. If
the mid point was located in a gene (NCBI human RefSeq mRNAs (NCBI_RefSeq) were
used to define the extent of a gene) the position closest to the midpoint and not in a gene
was identified. The flanking BAC-CGCs were then extended to this point, or in the case of
the first and last BAC-CGCs on a chromosome to the start or end coordinate of the

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chromosome. Thus all nucleotides in the bovine genome sequence were included in a block
and therefore the virtual sheep genome sequence is exactly the same length as the bovine
Btau3.5x genome sequence.
The order and orientation of the bovine genome assembly Btau3.5x-based sheep BAC-CGCs
in the vsg2.0 was determined on the location and organisation of the sheep linkage map
markers (Maddox, et al., 2001) mapped to the Btau3.1 genome and converted to the Btau3.5x
assembly using the Btau3.1 to Btau3.5x coordinate conversion chain file and the LiftOver
utility. Using Perl scripts the agp file (AGP_file_specification) was built and used to
generate the sequence of the virtual sheep genome assembly, the coordinate conversion
chain file (for use by the LiftOver utility), and the contig and scaffold tracks for the virtual
sheep genome browser (VSG).
Using the LiftOver utility and the Btau3.5x to virtual sheep genome coordinate conversion
chain file, the BES and BAC-CGC mapping coordinates, and any other features mapped to
the Btau3.5x bovine genome, were converted to the virtual sheep genome coordinates.
Features were also transferred from the Btau3.1 genome assembly by first converting to
Btau3.5x coordinates using the Btau3.1 to Btau3.5x coordinate conversion file and the
LiftOver utility and then converting from Btau3.5x to vsg2.0 coordinates. Other features
were mapped directly onto the virtual sheep genome using sequence alignment programs
such as BLAST and BLAT with the vsg2.0 DNA sequence.
3.1 Identification of problems with the Btau3.1 assembly of the bovine genome
The cow is the most closely related organism to sheep for which a genome assembly is
available. When this project was commenced, an early draft of the bovine genome assembly
Btau3.1 (Elsik, et al., 2009) was in the public domain. Since the sheep genome assembly
would be built comparatively on the bovine genome, and sheep sequence contigs from the
low coverage six animals at approximately 0.5 fold coverage each, were expected to be very
small, the accuracy of the bovine genome assembly would determine the accuracy of the
sheep assembly at all levels above that of the individual sequence contigs.
To assess the validity of this strategy the sheep BESs from the CHORI-243 library were
mapped to the Btau3.1 genome assembly to identify the extent of segments of conserved
synteny between the two genomes. The reader should keep in mind that the only BACs
counted as being in the same organisation in the comparison genome as in the source
genome (i. e. congruent) are the tail-to-tail BACs less than 200kb in length. Unexpectedly
large numbers of sheep BACs, more than 17% of the BACs with both ends mapped, had
both BESs positioned on the bovine Btau3.1 genome assembly within 200kb of each other,
but not in the expected tail-to-tail organisation, i. e. many BACs had their two BESs
mapped in the tail-to-head and head-to-head organisations (Table 1). In addition, large
numbers of BACs had both BESs positioned on the same chromosome, but more than
200kb apart, the outsize groups (Table 1). The average insert size of the BACs in the sheep
BAC library is 184kb (Dalrymple, et al., 2007).
Such a result would normally suggest a substantial number of intra-chromosomal
rearrangements between the sheep and cattle genomes. However, almost as many, more
than 14%, of bovine BACs were also not positioned as tail-to-tail BACs on the bovine
Btau3.1 genome assembly (Table 1). The organisation of sheep BACs at the locations of these
apparent rearrangements between the two genomes was compared with the organisation of


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bovine BACs at the same locations in the genomes. Frequently clusters of tail-to-head sheep
BACs overlapped with clusters of tail-to-head bovine BACs (Fig 1), suggesting that many
such occurrences were in fact due to an incorrect assembly of the bovine genome, not true
differences in the structures of the two genomes themselves. However, many clusters of tail-
to-head sheep BACs that did not overlap with tail-to-head bovine BACs were also observed
(Fig 1). These BACs probably represent rearrangements in the sheep genome relative to the
bovine genome.

Fig. 1. Segment of chromosome one of the bovine Btau3.1 genome assembly showing the
positions and orientations of sheep and cattle BACs. BCM genome assembly contigs are
coloured based on the human chromosome to which they have the highest scoring match.
The circles identify regions of likely inversion in the bovine and/or sheep genomes relative
to the Btau3.1 genome assembly.

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Genome Assembly Btau3.1 Btau3.1 Btau3.5x vsg2.0 vsg1.2
BAC origin cattle sheep cattle sheep sheep
tail-to-tail (%) 86.7% 82.7% 95.63% 94.0% 89.6%
tail-to-tail outsize 2.0% 2.6% 0.4% 0.8% 1.7%
tail-to-head 3.5% 4.4% 2.6% 2.1% 2.6%
tail-to-head outsize 5.2% 7.1% 0.9% 2.1% 4.4%
head-to-head 0.4% 0.4% 0.4% 0.2% 0.3%
head-to-head-outsize 2.1% 2.8% 0.1% 0.6% 1.4%
tail-to-tail (number) 67,352 47,818 82,765 95,757 84,624
breaks 13,192 19,151 27,829
unpaired 79,172 50,142 52,663
Table 1. Mapping of cattle and sheep BACs to assemblies of the cattle and virtual sheep
genomes.
3.2 Using cattle and sheep BACs to reorganise the Btau3.1 assembly of the bovine
genome
The first step in the generation of the virtual sheep genome was therefore to construct the
best approximation to the correct order of the bovine sequence contigs and scaffolds in the
bovine genome using the bovine and sheep BACs and comparative genomics. Initially, the
scaffolds in the bovine genome assembly (Btau3.1) were kept intact and scaffolds were
reordered and reoriented within bovine chromosomes to minimize the number of both cattle
and sheep BACs that were not in the tail-to-tail organisation. Then scaffolds apparently
assigned to the wrong chromosomes on the basis of the BAC-based links to other scaffolds
in the assembly were moved, including being inserted into gaps in other scaffolds guided by
the mapping of the BESs. Generally these moves were also supported by the mapping of the
sequence contigs to the human, dog and horse genomes (Fig. 2). In addition, scaffolds not
assigned to chromosomes in Btau3.1 were included in the assembly where BACs provided
unambiguous links. Finally, reordering and reorienting of contigs within the new set of
ordered and reoriented scaffolds was undertaken.
Given the size of the BACs and the variation in the length of the genomic DNA contained
within the BACs the correct position to insert many segments of the bovine assembly was
ambiguous based solely on the BAC-end data. Throughout this process, which was mainly
undertaken manually, the alignment of the bovine genome assembly contigs to the human,
dog and horse genome assemblies was used in making the final decision about where
exactly to insert or break scaffolds. In other words, a breakpoint between sequence contigs
in an assembly scaffold was chosen that was consistent with the cattle and sheep BES data
and the organisation of the human, dog and horse genomes (Fig. 2). Where conflicts
between the comparative genome assemblies occurred two out of three consistent
organisations were required. However, the integrity of sequence contigs was maintained
throughout the process, although evidence for chimeric sequence contigs was also identified
during the course of the analysis (data not shown).
To avoid ovinising the bovine genome at least one bovine BAC was required to support all
reorganisations, except reordering and reorienting scaffolds within chromosomes in cases
where the bovine BAC fingerprint map (Snelling, et al., 2007) also supported the


380

Fig. 2. Segment of chromosome one of the bovine Btau3.5x genome assembly showing the
positions and orientations of sheep and cattle BACs. BCM genome assembly contigs are
coloured and orientated based on the relevant species chromosome to which they have the
highest scoring match The UCSC chromosome colour scheme was used (Fujita, et al., 2011).
reorganisation. This process was undertaken reiteratively to resolve any errors introduced
or new links identified as the chromosome structures approached the most likely structure
of the bovine genome. This revised assembly of the bovine genome based on Btau3.1 was
named Btau.3.5x.

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In Btau3.5x the number of bovine assembly scaffolds was reduced from 3053 scaffolds
assigned to chromosomes in Btau3.1 to 537 super-scaffolds linked by the cattle and sheep
BACs. Of the chromosomally assigned scaffolds in Btau3.1, 974 scaffolds were inverted, and
683 scaffolds were split into 1720 pieces, of which 710 were inverted. 14 scaffolds were
moved to a different chromosome and 2192 scaffolds previously not assigned to
chromosomes were incorporated into the assembly. 104 of these scaffolds were split into 233
pieces. Coverage of the genome with scaffolds assigned to chromosomes increased from 2.4
Gb to 2.77 Gb. Even after this process it is likely that there remained a number of segments
of the bovine genome assembly which may not have been correctly assembled.

chromosome scaffold orientation integrity
1 BTA1.1 split
1 BTAUn.418 inv
1 BTAUn.728 inv
1 BTA1.2
1 BTA1.3
1 BTA1.4
1 BTAUn.1364 inv
1 BTAUn.208
1 BTA1.6 split
1 BTAUn.2125 inv
1 BTA1.6 split
1 BTAUn.1381
1 BTA1.5 split
1 BTAUn.1438 inv
1 BTA1.5 split
1 BTA1.7 split
1 BTAUn.3041
1 BTAUn.5341 inv
1 BTA1.7 split
1 BTA1.8
Table 2. The first twenty scaffolds of the bovine Btau3.5x assembly, scaffolds numbered
BTA1.* were assigned in numerical order to chromosome 1 of the bovine genome assembly
Btau3.1 build. Scaffolds numbered BTAUn.* were not assigned to a chromosome in the
bovine Btau3.1 build. inv indicates scaffolds inverted in the Btau3.5x genome build
relative to the Btau3.1 build, and split indicates scaffolds split in the Btau3.5x build
relative to the Btau3.1 build.


382
3.3 Integration of the positions of sheep BESs on the Btau3.5x, dog, horse and vsg1.2
genome assemblies
We then used the virtual genome strategy (Dalrymple, et al., 2007), integrating the separate
mapping of the sheep BESs to the original virtual sheep genome (v1.2), the dog and the
horse genome assemblies, to maximise the positioning of sheep BESs on Btau.3.5x. There
was little change in the human genome assembly over the course of the work and mapping
of the sheep BACs to the human genome was captured by using the virtual sheep genome
v1.2. Thus the virtual sheep genome version 2 was build on top of v1.2, rather than being a
completely de novo version. This approach, which uses much lower specificity BLAST
parameters, increased the number of sheep BACs able to be positioned on the bovine
genome substantially, from 47,818 (in the initial alignments) to 95,757 in the virtual sheep
genome, effectively doubling the coverage of the genome (Table 1). The number of sheep
BACs able to be positioned in the tail-to-tail organisation in a genome is a complex function
of the sequence coverage, assembly stage and evolutionary distance from the bovine
genome. The greater distance of the dog genome appears to be partially compensated for by
the more advanced state of the assembly used in this analysis. Very similar numbers of
BACs were mapped in the tail-to-tail organisation to the two genomes (Table 3) with similar
numbers of unique BACs (Table 4).

bovine horse dog vsg1.2
bovine 77,320
horse 49,225 60,971
dog 46,355 47,142 57,192
vsg1.2 62,889 56,636 54,503 84,624
Table 3. Tail-to-tail BACs within each dataset generated by independently mapping the
sheep BESs to each genome and in the intersections between each of the datasets.

genome including vsg1.2 excluding vsg1.2
bovine 10,550 20,171
horse 701 3,035
dog 211 2,063
vsg1.2 9,204 not applicable
Table 4. Tail-to-tail BACs unique to each dataset.
The high coverage and quality of the human genome assembly and the use of the
integration strategy presumably contributed to the large number of unique BACs in the tail-
to-tail organisation present in vsg1.2 (Tables 3 and 4). Over and above the newer assembly
of the bovine genome the inclusion of the mapping of the sheep BACs to the horse genome
assembly has the biggest impact on the number of BACs assigned and on the number of
BAC contigs, where fewer is better (Table 5). This is not surprising since, of the genomes
used, the horse is the most closely related species to the two ruminants.
Adding the horse mapping of the sheep BESs positions to the bovine mapping of the sheep
BESs positions increased the number of BACs mapped by 13,940, mainly by generating

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BACs with one end directly mapped to the bovine genome and other end mapped to the
bovine genome via the horse genome (Table 6). Subsequent addition of the BESs mapped via
the dog genome added many fewer BACs than adding the BESs mapped via the horse
genome (Table 6). The subsequent addition of the human genome data, incorporated in the
vsg1.2, added slightly more BACs than the addition of the dog genome (Table 4). Thus
including the dog genome had only a small impact on the improvement in the coverage of
the virtual sheep genome whereas the more distant, but the better assembled/higher
coverage, human genome was a useful addition to the virtual genome construction, but not
unexpectedly the biggest contributions came from well assembled genomes of closely
related species.

bovine horse dog
vsg1.2
total
bovine 76,251 76,251
horse 13,231 709 13,940
dog 1,911 112 45 2,068
vsg1.2 3,131 284 68 55 3,538
total 95,797
Table 5. Genomes providing mapping information for the sheep BACs mapped tail-to-tail in
the vsg2.0. Datasets were added in the order, bovine, horse, dog and vsg1.2.

Genome BACs positioned by number
Both BESs vsg1.2 80,146
Both BESs bovine 13,196
One BES bovine or vsg1.2, other BES horse or dog 2431
Both BESs horse 16
One BES horse, other BES dog 5
Both BESs dog 3
95,797
Table 6. Genomes used to position the BACs on the virtual sheep genome. Datasets were
added in the order, vsg1.2, bovine, horse, and dog.
In other words, building on top of vsg1.2 and the use of a higher quality assembly of the
bovine genome contributed a large number of new BACs with both ends positioned on the
bovine assembly (Table 5). A large group of BACs were positioned with one end using the
bovine or vsg1.2 position and the other using horse or dog. Very few BACs were positioned
solely using horse and/or dog positions (Table 6). On this basis further improvement of the
vsg would appear to be difficult and most likely to come from filling of gaps in the bovine
genome sequence itself.
Based on the mapping of the sheep BACs to the reorganised bovine genome assembly 943
blocks of conserved synteny, defined by overlapping sheep BACs, were identified between
the sheep and cattle genomes (Table 7). Assuming a genome size of 3Gb, the blocks had an
average length of just over 3Mb. Although initially disappointing, even in the bovine
genome assembly 537 BAC-based super-scaffolds were required to cover the complete


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genome. The comparison of the number of blocks of conserved synteny identified across the
different combinations of datasets demonstrates that the inclusion of additional species
beyond the horse has a much greater impact on the reduction in the number of blocks of
conserved synteny than it has on the total number of BACs positioned tail-to-tail. Only a
25% increase in the number of BACs, but a 56% decrease in the number blocks of conserved
synteny, i. e. on average every block of conserved synteny defined based on the mapping of
BACs to the bovine genome has been extended to include one adjacent block of conserved
synteny.

genomes Sheep BAC contigs
dog 2,146
horse 1,470
bovine 1,411
bovine + dog + vsg1.2 1,299
bovine + horse + dog + vsg1.2 943
Table 7. Building the virtual sheep genome.
3.4 Remaining ambiguities in the build of the bovine genome
Since there were many occasions on which there was no unambiguous basis on which to
identify the correct break points in the bovine genome assembly a large number of probable
inversions identified by BACs remained in the final version of the bovine genome. Most of
these inversions were also supported by sheep BACs (Fig 2). In addition, whilst potentially
chimeric bovine genomic sequence contigs were identified during the reassembly process,
their structure has not been changed in Btau3.5x.
3.5 Construction of the virtual sheep genome (vsg2.0)
The sheep markers (sheep map version 4.7) were used to reorganise the bovine genome
assembly into the vsg. In the main this involved renumbering of the bovine chromosomes,
with five inverted chromosomes (or segments of chromosomes), four chromosome fusions
and a single chromosome breakage (Table 8). Reordering of the segments of the bovine
genome defined by the BAC comparative genome contigs (CGCs) was undertaken on four
chromosomes, 7, 12, 13 and X. Apart from the X chromosome, these were local changes and
involved a small number of BAC CGCs covering a small region of the genome. Given the
variation in the size of BACs, and the lack of comparative data from other genomes for
species specific breaks, the boundaries of such breaks could not be unambiguously
identified with the data currently available. Thus no attempt was made to resolve the small
potential sheep specific rearrangements within chromosomes where the break points were
ambiguous and there was not sufficient marker evidence to support a change in the
organisation (Fig 3).
The vsg 2.0 has been used in a number of analyses of the genome organisation of sheep and
in general a high level of congruence with maps determined using other approaches has
been observed (Drogemuller, et al., 2008; Wu, et al., 2008; Goldammer, et al., 2009c; Wu, et
al., 2009), although the vsg 2 X chromosome build appears to contain a number of significant
discrepancies (Goldammer, et al., 2009a; Goldammer, et al., 2009b).

385
Sheep chromosome Cattle chromosome
OAR1 BTA3 (inv) + BTA1
OAR2 BTA8 (inv) +BTA2
OAR3 BTA11 (inv) + BTA5
OAR4 BTA4
OAR5 BTA7
OAR6 BTA6
OAR7 BTA10
OAR8 BTA9 (part)
OAR9 BTA9 (part, inv) +BTA14
OAR10 BTA12
OAR11 BTA19
OAR12 BTA16
OAR13 BTA13
OAR14 BTA18
OAR15 BTA15
OAR16 BTA20
OAR17 BTA17
OAR18 BTA21
OAR19 BTA22
OAR20 BTA23
OAR21 BTA29
OAR22 BTA26
OAR23 BTA24
OAR24 BTA25
OAR25 BTA28
OAR26 BTA27
OARX BTAX (inv)
Table 8. High level comparison of the sheep and cattle genomes based on virtual sheep
genome analysis.
3.6 Construction of the virtual sheep genome (vsg2.0) genome browser
The cattle and sheep BAC and BES locations are displayed on the chromosome overview
track of the virtual sheep genome browser (VSG) allowing a quick assessment of the quality
of the assembly to be made (Fig 3). In addition, the sheep virtual genome assembly was
annotated with the locations of the sheep markers, SNPs on the 1536 pilot sheep SNP chip
(Kijas, et al., 2009) and the Illumina Ovine SNP50 BeadChip, and human and bovine mRNA
RefSeqs downloaded from the NCBI (NCBI_RefSeq).


386

Ovine Tail-Tail Outsize BACs

Ovine Tail-Head BACs

Ovine Tail-Head-Outsize BACs

Ovine Head-Head BACs

Ovine Head-Head-Outsize BACs

Ovine unpaired BESs

Bovine Tail-Tail Outsize BACs

Bovine Tail-Head BACs

Bovine Tail-Head-Outsize BACs

Bovine Head-Head BACs

Bovine Head-Head-Outsize BACs

Sheep Markers

1536 Pilot SNPs

Fig. 3. Overview of chromosome OAR23 from the vsg v2 browser, displaying ovine and
bovine BAC mapping, sheep linkage map markers and Pilot SNP Chip SNPs.

387

Fig. 4. A 5 Mb segment of the vsg 2 genome assembly of chromosome 23 showing the
positions and orientations of sheep BACs and other tracks.
4. Conclusion
The new vsg2.0 is a significant improvement over vsg1.2, built on the human genome
framework. Clearly using the genome from a closely related species and allowing the data
from the species of interest to direct the process has an advantage over a very well
assembled, but more distant genome. At the low resolution level down to the level of the
BACs the sheep genome has a very high level of overall conserved synteny with the bovine
genome structure. A number of regions of ambiguity remain, but many of these are in


388
regions of ambiguity of the assembly of the bovine genome and therefore await further
refinement of the bovine genome assembly, or a predominantly de novo assembly of the
sheep genome. However, overall it is clear that the vsg 2 makes a robust framework to
assemble the large number of short contigs expected from the sequencing of the sheep
genome (Archibald, et al., 2010).
Two assembled genomes from closely related species is probably the optimal balance
between analysis complexity and benefit, with inclusion of a more distant, but much better
assembled genome, if the genomes of closely related species are not well assembled. Thus
the methods that we have described are very broadly applicable.
5. Acknowledgement
The authors would like to thank the members of the International Sheep Genomics
Consortium (ISGC_website) in particular Jill Maddox, John McEwan and James Kijas for
useful discussions. The authors also gratefully acknowledge the early pre-publication access
under the Fort Lauderdale conventions to the draft equine genome sequence provided by
the Broad Institute and to the draft bovine genome sequence provided by the Baylor College
of Medicine Human Genome Sequencing Center and the Bovine Genome Sequencing
Project Consortium. This work was partly funded by SheepGenomics (a joint venture of
Meat and Livestock Australia and Australian Wool Innovation). The work was undertaken
as part of the development of sheep genomics tools by the ISGC.
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19
Basidiomycetes Telomeres
A Bioinformatics Approach
Luca Ramrez, Gmer Prez, Ral Castanera,
Francisco Santoyo and Antonio G. Pisabarro
Genetics and Microbiology Research Group, Public University of Navarre, Pamplona,
Spain
1. Introduction
1.1 The telomere: A complex nucleoprotein complex with a broad range of functions
The telomeres (from the Greek tlos far and -meros part) are the genetic structures found at the
physical ends of linear chromosomes. They are nucleoprotein complexes composed of DNA
repeats and a myriad of telomere and non-telomere associated proteins aimed to protect the
ends of eukaryotic chromosomes from being recognized as double strand breaks, and to avoid
chromosome end degradation by nucleases and non-canonical chromosome end fusions. Thus,
telomeres are essential for chromosome integrity (Hande, 2004; Paeschke et al., 2010; Zakian,
1995). The fascinating story about telomere biology comes from the pioneering work of
Elizabeth H. Blackburn who discovered that Tetrahymena telomeres consisted of a short DNA
sequence motif that was repeated several times at the chromosomal end (Blackburn & Gall,
1978). This pattern is conserved in lower eukaryotes and in mammalian cells (Greider, 1998).
Notable exceptions are Drosophila and some other dipterans, which instead possess tandem
arrays of retrotransposons at their chromosome ends (Abad et al., 2004).
Telomere DNA consists of tandem arrays of short repeated sequences forming a cap.
Telomere length is species-specific and small cell type variations were observed. For
reviews, see Fisher & Zakian, (2005) and Sanchez-Alonso & Guzman (2008). The basic
telomere DNA repeat unit is the hexamer TTAGGG in which the strand running 5 3
outwards the centromere is usually guanine-rich. This G-rich strand protrudes its
complementary end and bends on itself to form a telomere DNA loop (T-loop) (Griffith et
al., 1999) which protects the structure from being recognized as a double-stranded break by
sequestering the 3-overhang into a high order DNA structure. The G-rich strand also serves
as an anchor for a telomere-dedicated reverse transcriptase, called telomerase, that
compensates for the inability of DNA polymerases to replicate the 5 ends of linear
chromosomes (Blackburn & Gall, 1978). The telomerase binds the G-rich strand by
complementary pairing of the protruding DNA sequence to the telomerase RNA subunit
and, as a result, telomerase elongates the overhang by adding telomere sequence repeats
(Masutomi et al., 2003; Morin, 1989; Zhao et al., 2009). The T loop structure is maintained by
a complex of telomere and non-telomere proteins called shelterins which repress the DNA
repair machinery at telomeres, and regulate telomere length (for review see de Lange, 2005;
Palm & de Lange, 2008; Rhodes et al., 2002; Vega et al., 2003; Zhao et al., 2009). The shelterin


394
complex is formed by a core of six proteins including the Myb-type homeodomain TRF
proteins in mammals, Rap1 in Saccharomyces cerevisiae and Taz1 in Schizosaccharomyces pombe
which bind the duplex form of the telomere repeats, the OB-fold containing protein POT1 in
mammals and S. pombe and Cdc13 in S. cerevisiae which bind the single-stranded telomere
3overhang and by other proteins associated via proteinprotein interactions with them
(Rhodes et al., 2002; Vega et al., 2003; Zhao et al., 2009). This shelterin complex is
evolutionary conserved although some differences between species appear in protein
numbers and in its higher order structure (Linger & Price, 2009). The G-rich single stranded
telomere tail is also able to form a secondary DNA order structure resulting from intra and
intermolecular G-quadruplex (Fry, 2007; Maizels, 2006). G-quadruplexes are stacked
associations of G-quartets, which are themselves planar assemblies of four Hoogsteen-
bonded guanines, with the guanines derived from one or more nucleic acid strands (De Cian
et al., 2008; Johnson et al., 2008). These structures have been observed in lower eukaryotes
(Paeschke et al., 2008; Paeschke et al., 2005; Schaffitzel et al., 2001) and have the potential to
regulate telomerase activity (Oganesian et al., 2006; Zahler et al., 1991).
Secondary DNA structures, G-quadruplex structures and T-loop may contribute to telomere
function but pose an obstacle for semi-conservative and telomerase-mediated replication, a
problem which should be solved to avoid telomere shorten. Telomeres become shortened
during every cell cycle due to incomplete replication of the lagging strand (the so called
end replication problem) resulting in cumulative telomere attrition during aging. In
addition, a loss of telomere DNA occurs due to post-replicative degradation of the 5 strand
that generates long 3 G-rich overhangs (Wellinger et al., 1996; Wellinger et al., 1993). In
most species, the loss of telomere DNA is counteracted by the action of telomerase that
carries its own RNA template coding for the telomere repeat sequence (Chan & Blackburn,
2004). The complementary C-rich strand is then synthesized by conventional RNA-primed
DNA replication (Gilson & Geli, 2007; Verdun & Karlseder, 2007). Following replication, the
telomeres created by the synthesis of the leading strand are either blunt-ended or left
carrying a small 50 bp overhang whereas those created by the lagging-strand synthesis have
a 3 overhang with a length determined by the position of the outermost RNA primer (de
Lange, 2009). This fact supports the importance of the telomerase activity for the genome
integrity. When telomeres reach a critical minimal length they become uncapped. This leads
to a permanent cell cycle arrest (termed cellular senescence) or to apoptosis, depending on
the cellular context in which the uncapping occurs (Aubert & Lansdorp, 2008; Blasco, 2005).
Extreme telomere shortening leads to chromosome instability, end-to-end fusions, and
checkpoint-mediated cell cycle arrest and/or apoptosis (reviewed in Aubert & Lansdorp
(2008) and in Shore & Bianchi (2009). The whole processes are related in mammals not only
to aging, but also to several age associated diseases such as tumorigenesis, coronary artery
disease, and heart failure (Donate & Blasco, 2011; Ogami et al., 2004; Sherr & McCormick,
2002; Starr et al., 2007). In addition to the role of telomerase in maintaining telomere length,
it has been described that homologous recombination (HR) constitute an alternative method
(ALT alternative lengthening of telomeres) to maintain telomere DNA in telomerase-
deficient cells with telomeres highly heterogeneous in length. This mechanism was
described in S. cerevisiae and consists in two pathways depending on different
recombination proteins that use different telomere sequences as substrates for
recombination. Cancer and immortalized cells can utilize the ALT mechanism to maintain
telomere length (Lundblad & Blackburn, 1993; Teng et al., 2000; Teng & Zakian, 1999).

Basidiomycetes Telomeres A Bioinformatics Approach

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Telomeres were considered as regions where the transcription of structural genes is
repressed (Mondoux & Zakian, 2005), although it has been recently reported that telomere
repeats and subtelomere regions can be transcribed (Azzalin et al., 2007; Luke & Lingner,
2009; Luke et al., 2008; Schoeftner & Blasco, 2008). The telomere transcribed region, called
TERRA (telomere repeat-containing RNA), forms an integral component of telomere
heterochromatin, and produces non-coding G-rich RNAs transcribed from the telomere C-
rich strand in mammals and fungi (Azzalin et al., 2007; Luke et al., 2008; Sanchez-Alonso &
Guzman, 2008; Schoeftner & Blasco, 2008). TERRA transcription occurs at most or all
chromosome ends and it is regulated by RNA surveillance factors and in response to
changes in telomere length. The accumulation of TERRA at telomeres can also interfere with
telomere replication (Azzalin & Lingner, 2007; Luke & Lingner, 2009; Schoeftner & Blasco,
2009).
The particular sequence organization of telomere makes difficult its genetic mapping and
sequencing due to they are cloning recalcitrant and underrepresented in mapping and final
assembled genomes. Consequently, their cloning and characterization must be made by
dedicated molecular and bioinformatics strategies (Perez et al., 2009; Sanchez-Alonso &
Guzman, 2008).
1.2 Subtelomere chromosome regions
Human subtelomere chromosome regions contain complex and dynamic stretches of DNA
which, together with their associated proteins, are essential for genome stability and proper
chromosome replication (Riethman et al., 2005). Subtelomere DNA repeats are a complex
region of variable size segmentally duplicated containing low copy DNA repetitive tracts
adjacent to the telomere. These duplications could be found only at the subtelomere regions,
although it is common to find them also at pericentromeric and interstitial chromosomal loci
(Riethman et al., 2001).
In humans, subtelomere DNA regions are operationally defined as the terminal 500 Kbp of
each euchromatic chromosome arm. These regions contain subtelomere repeats (Srpts),
segmental duplications, satellite sequences, and internal (TTAGGG)
n
-like sequences
(Riethman et al., 2004). The organization of the subtelomere region is structurally conserved
across eukaryotes (Anderson et al., 2008; Brown et al., 1990a; Brown et al., 1990b; Chan &
Tye, 1983a; 1983b; Flint et al., 1997a; Flint et al., 1997b; Karpen & Spradling, 1992; Levis,
1993; Louis, 1995; Louis & Borts, 1995; Mefford & Trask, 2002; Pryde et al., 1997; Pryde &
Louis, 1997; Walter et al., 1995; Wilkie et al., 1991). This region, susceptible to
hypermethylation has been recently shown to have a central function in mammalian
telomere-length homeostasis (Blasco, 2007). Subtelomere repeats are characterized by their
high level of polymorphism among different chromosome ends and among individuals of
the same species. This polymorphism, possibly indicative of a quick and dynamic sequence
turnover, leads to a lack of relationship among subtelomere repeats across species.
Nonhomologous or ectopic exchange between subtelomere regions of different
chromosomes has been reported as a possible reason of polymorphisms in both yeast and
humans (Linardopoulou et al., 2005; Louis & Haber, 1990; Mefford & Trask, 2002). In
humans, there are more re-arrangements at the sub-telomere regions than in the rest of the
genome. This is also true in some lower eukaryotes such as Plasmodium falciparum (Freitas-
Junior et al., 2000), Magnaporthe oryzae (Rehmeyer et al., 2006) and Neurospora crassa (Wu et
al., 2009), among others. In all these cases genes involved in niche adaptation (species-


396
specific genes) were found in the subtelomeric regions. It could be that the high evolutive
potential of the subtelomeric regions were used by these organisms to create variability
aimed to avoid the detection by the host. In fungi the genes more frequently found in
subtelomere regions are transposons, telomere-linked RecQ helicases, clusters of secondary-
metabolites, cytochrome oxidases, hydrolases, molecular transporters, and genes encoding
secreted proteins (Perez et al., 2009). These genes could undergo transcriptional silencing
(subtelomere silencing) due to its close proximity to telomeres.
1.3 Interstitial telomere repeats
The various chemical modifications occurring at the amino terminal end of the histones
affect the structure of chromatin and help establishing the functional and structural domains
known as euchromatin and heterochromatin. Euchromatin is an open form of chromatin
that allows transcription factors access to and transcriptionally activate their target genes. It
is largely occupied by housekeeping genes, condensed during metaphase and decondensed
during interphase. Heterochromatin, on the contrary, differs from euchromatin in that it is
condensed during interphase.
Heterochromatin has been often said to be poor in genes and mainly constituted by
repetitive DNA sequences. Moreover, since it is highly condensed and inaccessible to
transcription factors, heterochromatin is generally transcriptionally silent (Hernandez-Rivas
et al., 2010). Heterochromatin appears as blocks spread over the chromosomes when they
are stained with Giemsa dye. The molecular analysis of heterochromatic blocks reveals
sequences similar to the telomere repeats that are called in this case interstitial telomere
repeat sequences or ITRs. These sequences include those repeats located close to the
centromere and those found at interstitial sites, i.e., between the centromere and the
telomeres (Meyne et al., 1990; Slijepcevic et al., 1996). ITRs were described in plants, animals
and humans (Bolzan & Bianchi, 2006; Uchida et al., 2002; Welchen & Gonzalez, 2005). At the
chromosome level, ITRs can be detected either by using the Fluorescence in situ
hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) pan-telomere
probe (i.e., a probe that identifies simultaneously all of the telomeres in a metaphase cell), or
by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer
complementary to the telomere DNA repeated sequence (Bolzan & Bianchi, 2006).
The length and the locations of the heterochromatic blocks in chromosomes are variable
(Azzalin et al., 2001; Faravelli et al., 1998; Weber et al., 1990) as well as their origin.
However, the presence of ITRs in the heterochromatic blocks is interpreted as the result of
tandem telomeretelomere fusions during evolution (Hastie & Allshire, 1989; Holmquist &
Dancis, 1979; Meyne et al., 1990) or the insertion of telomere DNA within genome unstable
sites (recombination hotspots) during the repair of double strand DNA breaks (DSB)
(Azzalin et al., 2001). The presence of some relatively small ITRs flanked by unstable AT-
rich DNA sequences could support this last hypothesis (Faravelli et al., 2002). On the other
hand, telomere associations and fusions are common cytogenetic findings that have been
implicated in the initiation of chromosome instability and tumorigenesis (Callen & Surralles,
2004; Murnane & Sabatier, 2004; Soler et al., 2005). Telomere fusions are the result of
telomere dysfunction due to attrition of chromosome ends (Maser & DePinho, 2004). They
are usually found in repair- and/or telomerase-deficient cells (Bailey et al., 1999; Blasco et
al., 1997; Hande, 2004; Hande et al., 1999; Lo et al., 2002; Samper et al., 2000) with a variety
of mutations affecting telomere function, including those occurring in proteins of the


397
shelterin complex (Bailey et al., 1999; Goytisolo et al., 2001). Mammal cells show a high
frequency of telomere fusions (end-to-end) and chromosome instability (Bailey et al., 2004;
Bailey et al., 1999; Espejel et al., 2002; Hsu et al., 2000; Smogorzewska et al., 2002; Takai et al.,
2003; van Steensel et al., 1998). The FISH technique allows the identification of metacentric-
submetacentric and acrocentric-telocentric chromosome telomere fusions also known as
Robertsonian-like configurations (Al-Wahiby et al., 2005; Hande, 2004). The occurrence of
telomeretelomere associations has been suggested to play a role in nuclear organization
(Nagele et al., 2001). In fact, telomere associations were seen in metaphases of human cells
with shorten telomeres suggesting that a minimal telomere length is required for a proper
chromosome function during mitosis.
2. Fungal telomeres - A bioinformatics approach
The basic and conserved telomere unit sequence in most filamentous fungi is TTAGGG. This
sequence has been described in Aspergillus nidulans (Bhattacharyya & Blackburn, 1997),
Beauveria bassiana (Padmavathi et al., 2003), Botrytis cinerea (Levis et al., 1997), Cladosporium
fulvum (Coleman et al., 1993), Fusarium oxysporum (Inglis et al., 2000), Glomus intraradices
(Hijri et al., 2007), Magnaporthe grisea (Gao et al., 2002), Metarrhizium anisopliae (Inglis et al.,
2005), N. crassa (Wu et al., 2009), Pestalotiopsis microspora (Long et al., 1998), Pleurotus
ostreatus (Perez et al., 2009), Pneumocystis carinii (Keely et al., 2001), and Ustilago maydis
(Sanchez-Alonso & Guzman, 2008). However, variations of this sequence can be found in
other fungi such as A. oryzae that has dodecanucleotide telomere repeats (Kusumoto et al.,
2003) Incomplete and imperfect telomere units have been reported in: A. oryzae, Candida
albicans, Kluyveromyces lactis, S. cerevisiae and S. pombe.
Two DNA sequence domains can be found adjacent to the telomere repeats. One of them,
distal, is placed next to the telomere and contains tandem repeat motifs. The other,
proximal, is interstitial, contains less repeated sequences and ferries clusters of related genes
(Pryde et al., 1997). In several fungi, it has been observed an increased number of proteins
involved in interactions with the environment coded for by genes mapping close to the
telomeres. These genes are called contingency genes, they are dispensable for survival and
are highly variable in populations. The accumulation of these genes near the telomeres is a
strategy that allows fungi to afford new environments. In fact, it has been observed in S.
cerevisiae that the genes located near the telomeres display variation in gene amplification
and/or expression depending on the growing niche of the yeast. Some of these genes belong
to the PAU family, the largest gene family in S. cerevisiae (23 members), whose regulation
depends on the environmental growing conditions (anaerobiosis) (Rachidi et al., 2000).
Other telomere associated genes in S. cerevisiae are MAL and MEL that participate in maltose
and melibiose fermentation, used in the baking and brewing industries (Gibson et al., 1997;
Teunissen & Steensma, 1995), and FLO that encodes cell-wall glycoproteins which
participate in the regulation of cellular adhesion (Gibson et al., 1997; Halme et al., 2004;
Teunissen & Steensma, 1995). Similarly, the TLO family unique in C. albicans consists of 15
members present on every chromosome, 14 of which are located at chromosome ends.
Genome comparisons between C. albicans and Candida dublinienesis showed that the
principal disparity in gene content between both species resides in the lack of the TLO genes
in this last one. CdTLO1 null strains show a major reduction in hyphal formation in
response to serum that can be reversed by complementation with either of two C. albicans
TLO genes (Jackson et al., 2009).


398
In human parasites it has been described several families of well characterized or putative
virulence factors at chromosome ends. In Candida glabrata (De Las Penas et al., 2003) nearly
24 adhesin encoding genes were located at telomere regions, and, in P. carinii clusters of
major surface antigen genes have been bioinformatically predicted at every chromosome
end (Keely et al., 2005). A similar situation was reported in P. falciparum and Trypanosoma
brucei where families of antigen surface proteins inducing immune responses are encoded in
telomere regions as part of a pathogens mechanism to amplify and diversify surface antigen
genes to avoid host recognition, as it has been hypothesized (Barry et al., 2003).
To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses,
Farmans group analyzed and characterized the telomere organization in the rice blast
pathogen M. oryzae (Rehmeyer et al., 2006) and in N. crassa (Wu et al., 2009). The molecular
and bioinformatics approach allowed them to identify 14 chromosomes in both of them. In
M. oryzae, the analysis of these sequences reveals the presence of a clearly defined distal
subtelomere domain that contains a telomere-linked helicase (TLH) gene. No gene
duplication near the chromosome termini is observed. Thus it is impossible to detect a
proximal subtelomere domain. The sequenced N. crassa genome (Galagan et al., 2003)
contains very little intact, duplicated DNA due to the repeat-induced point mutation (RIP)
process (Selker, 1990). This situation made unlikely that N. crassa would possess intact
subtelomere domains or terminal gene duplications. The search for tandem repeats at the
ends of some chromosomes whose sequences extend out of the telomere repeats shows the
lack of conserved subtelomere tandem repeats. A similar situation is observed when the
sequences immediately adjacent to the TTAGGG repeats were compared between strains.
Consistent with the absence of distinct subtelomere elements, N. crassa lacks the TLH genes
that are present in the subtelomere regions of diverse fungi (Gao et al., 2002; Inglis et al.,
2005; Louis & Haber, 1992; Mandell et al., 2005; Perez et al., 2009; Rehmeyer et al., 2006;
Sanchez-Alonso & Guzman, 1998). As in other fungi, the terminal regions of N. crassa
chromosomes ferry genes related to secondary metabolism such as a monooyygenase
(CYP450), a FAD-binding domain containing protein, a second CYP450, an O-
methyltransferase, a polyketide synthase, a major facilitator superfamily efflux pump, a
putative transcription factor, and an oxidoreductase. Apart from the clusters of secondary
metabolite genes, there were no genes overrepresented in the chromosome ends, except
those predicted to code for enzymes related to plant cell-wall degradation activity.
In basidiomycetes, despite the genomes of an ever growing number of genera involved in
lignin degradation, enzyme production, and bio pulping have been sequenced and
annotated, the information on the characteristics of their telomere and sub-telomere regions
is rather limited. As it was discussed above, this is due to the particular characteristic of
telomeres that make them refractory to cloning and difficult to sequence and assemble in
whole genome sequencing projects. A consequence of the difficulty in cloning telomeres is
that, for most organisms, there is limited information on the organization of chromosome
ends. Data about basidiomycete telomeres are available for U. maydis (Sanchez-Alonso &
Guzman, 1998), M. anisopliae (Inglis et al., 2005) and P. ostreatus (Perez et al., 2009). In the
three cases, the conserved telomere repetitive unit is TTAGGG, and the number of tandem
repetitions varies among species: 37 times in U. maydis, from 18 to 26 in M. anisopliae, and
from 25 to 150 in P. ostreatus. In all these cases, genes coding for RecQ helicases have been
found adjacent to the telomere regions.
In P. ostreatus, a lignin degrader edible mushroom, the analysis of telomere organization
was carried out with a combination of genetic, molecular, and bioinformatics tools. This


399
approach allowed our group to map 19 out of the 22 chromosome ends expected in its
linkage map (Perez et al., 2009), as well as to study the telomere adjacent regions. Similar
strategies have been described by different authors (Rehmeyer et al., 2006; Sanchez-Alonso
& Guzman, 2008; Wu et al., 2009). The search for telomere regions was performed in whole
genome sequence draft assemblies. These preliminary genome sequence versions appeared
as incomplete, contained some genes truncated and were misassembled. The rationale for
using them is that the genome final assembling strategies very often alter or eliminate the
telomere and subtelomere repetitive sequences of the fully assembled genomes. We used the
open-access Tandem Repeats Finder program (TRFp, http://tandem.bu.edu/trf/trf.html) to
screen for repetitive telomere sequences in more than 6,200 contigs of the 4X coverage draft
sequence assembly of P. ostreatus PC15 produced by the Joint Genome Institute
(http://genome.jgi-psf.org/PleosPC15_1/PleosPC15_1.home.html). The TRFp locates and
displays tandem repeats in a DNA sequence file submitted in FASTA format without need
of specifying the repetitive pattern, its size or any other parameter. The TRFp output
consists of two files: a repeat table and an alignment. The repeat table contains information
about each repeat, its size, copy number, nucleotide content and location. Clicking on the
location indices for this tables entries opens a second web browser that shows an alignment
of the copies against a consensus pattern. TRFp is a very fast program that permits the
analysis of up to 5 Mb sequence length. Repeats with pattern size in the range from one to
2000 bases could be detected (Benson, 1999). We identified, in each telomere sequence
containing scaffold, the filtered gene models computer and manually annotated within 50
Kbp of the telomere repetitive sequence. This was done by manual inspection of each one of
the predicted genes and using them as query in the non-redundant NCBI gene database
using the BlastX program (Altschul et al., 1997). The BlastX results were considered
significant if their expected value (e-value) was <e-20. The identified protein sequences were
also used to query the online Pfam database using default parameters (Bateman et al., 2004).
A similar approach has also been used by Wu et al. (2009) to search for subtelomere regions
containing gene in N. crassa. This type of multiple analysis (genetic, molecular, and
bioinformatics) allows the characterization of most of the P. ostreatus telomeres as well as
several subtelomere regions that show high nucleotide similarity. The highly polymorphic
subtelomere region of P. ostreatus chromosome six contains genes similar to those described
in other eukaryotic organisms (RecQ helicases), apart from a species-specific laccase gene
cluster (six out of 12 genes annotated in the genome (Perez et al., 2009).
In conclusion, the assemblage of telomere regions by bioinformatics strategies is a powerful
tool to determine the arrangement of genomes in putative linkage groups in species with no
genetic maps available, to establish synteny among different basidiomycete genomes, and to
determine the presence of genes and gene clusters conserved in the subtelomere regions of
different genomes.
2.1 Analysis of the basidiomycetes telomere regions
In the following sections we will review the composition and structure of the telomere and
subtelomere regions of the different basidiomycetes using the bioinformatics approach
described above. We will use the genome sequence data that are publicly available at the
Joint Genome Institute (DOE-JGI, http://www.jgi.doe.gov/). This institute has been
developing an intensive sequencing effort on different fungi related with the biological
lignocellulose degradation. Lignin is a complex recalcitrant macromolecule that hinders the
access of enzymes to cellulose. The enzymatic removal of lignin will permit the access to this


400
large carbon reservoir for its use in different energy-related applications. There are two
types of fungi according to their strategy for making cellulose accessible: white rot fungi
degrade lignin, and brown rot fungi minimally modify the lignin and attack the cellulose
using a different chemical approach (Lundell et al., 2010; Martinez et al., 2005; Ruiz-Duenas
& Martinez, 2009). Among all the sequenced basidiomycetes, we will concentrate here on
the white rot degraders Ceriporiopsis subvermispora, Phanerochaete chrisosporium and P.
ostreatus; the brown rot Postia placenta and the tree pathogen Heterobasidion annosum.
2.1.1 Analysis of the telomere regions of Ceriporiopsis subvermispora B
C. subvermispora is a white rot basidiomycete that rapidly depolymerizes lignin with
relatively little cellulose degradation when growing on wood (Martinez et al., 2005). The
chromosome number of this species has not been conclusively determined. The JGI has
sequenced the strain B. Two type of sequence data (un-assembled reads and assembled
scaffolds unmasked) were screened for the presence of telomere sequences in the genome of
C. subvermispora: 297,269 unassembled and 740 assembled scaffolds. In 207 unassembled
scaffolds, between five and 23 tandem repeats of the telomere motif TTAGGG were found.
In most cases (82 % of these unassembled scaffolds) 17 and 19 repeats of the motif were
found, and the mean repeat number was 18.7. On the other hand, 187 scaffolds carried
between seven and 22 repeats of the telomere complementary sequence CCCTAA. In this
case, the modal repetition number varied between 17 and 19 (84% of the scaffolds) and the
mean number was 19.2. Taken together these data suggest that the average number of
telomere repeats in C. subvermispora is 19.0.
The unmasked analysis of 740 assembled scaffolds revealed that 42 of them contained
telomere sequences: 22 harbored the TTAGGG sequence and 20 the complementary
CCCTAA. The telomere region TTAGGG was placed at the bottom (3 telomere) end of the
chromosome in 21 out of the 22 scaffolds. An exception to this was observed in scaffold 3.
The sequence was located at an interstitial position. The telomere region CCCTAA was
placed at the upper (5 telomere) end of the chromosome in 19 out of 20 scaffolds. As it was
described above, this sequence was also placed at an interstitial location in scaffold 3. This
suggest a missassembling scaffold 3 because both the direct TTAGGG
21
(TTAGGG, scaffold
3: 1205371-1205497) and the reverse CCCTAA
21
(CCCTAA, scaffold 3: 1206763-1205497)
telomere sequences have been found flanking a 1265 bp gap 3 (Fig. 1).

Fig. 1. Location of interstitial telomere sequences in scaffold 3 of the genome sequence of C.
subvermispora.
Scaffold 3: 2650498 bp
5
(5 TTAGGG 3)
21
scaffold_3:1205371-1205497
(5 CCCTAA 3)
21
scaffold_3:1206763-1206881
GAP(1264 bp)
scaffold_3:1205498-1206762
3
5
(5 TTAGGG 3)
21
scaffold_3:1205371-1205497
(5 CCCTAA 3)
21
scaffold_3:1206763-1206881
(5 TTAGGG 3)
21
scaffold_3:1205371-1205497
(5 CCCTAA 3)
21
scaffold_3:1206763-1206881
GAP(1264 bp)
scaffold_3:1205498-1206762
3


401
The telomeres found in scaffolds 5 and 7 have a complex structure. The telomere of scaffold
5 contained 22 and 20 copies of the CCCTAA sequence separated by a gap of 193 bp. In the
case of scaffold 7, 20 and 18 copies of this sequence appeared separated by a gap of 1680 bp
(Fig. 2). We presume that these particular structures could reflect misassemblages of the
telomere sequences in both scaffolds.

Fig. 2. Location of telomere regions in scaffolds 5 and 7 of the genome sequence of C.
subvermispora.
In summary this analysis has allowed the identification of 42 telomere repeat containing
regions. 22 out of them fit with direct telomere sequences (TTAGGG) present in 22 scaffolds
and 22 reverse telomere regions (CCCTAA) present in 20 scaffolds. Taking into account that
the C. subvermispora strain sequence was a dikaryon, these results suggest that the
Ceriporiopsis genome would consist of 11 chromosomes. Because scaffold 7 showed telomere
repeats at both ends, the sequence contained in this scaffold would be the only fully
assembled chromosome of C. subvermispora.
2.1.2 Analysis of the telomere regions of Phanerochaete chrysosporium strain RP78
P. chrisosporium is a model white rot basidiomycete that has been extensively used because
of it interest as lignin degrader (Kersten & Cullen, 2007; Tien, 1987). The draft genome of the
homokaryotic strain of P. chrysosporium strain RP78 was assembled into 232 scaffolds and
contains 35.1 Mbp of non-redundant sequences (Martinez et al., 2004). 90% of the assembly
was found in 21 scaffolds, while 50% was found in eight scaffolds larger than 1.9 Mbp.
(5 CCCTAA 3)
22
scaffold_5:1-132
(5 CCCTAA 3)
20
scaffold_5:979-1098
3 5
GAP (193 bp)
scaffold_5:785-978
Interstitial (651 bp)
scaffold_5:133-184
(5 CCCTAA 3)
20
scaffold_7:1-120
(5 CCCTAA 3)
18
scaffold_7:2437-2545
GAP (1680 bp)
scaffold_7:756-2436
3 5
Intertitial (634 bp)
scaffold_7:121-755
(5 CCCTAA 3)
22
scaffold_5:1-132
(5 CCCTAA 3)
20
scaffold_5:979-1098
3 5
GAP (193 bp)
scaffold_5:785-978
scaffold_5:133-184
(5 CCCTAA 3)
22
scaffold_5:1-132
(5 CCCTAA 3)
20
scaffold_5:979-1098
(5 CCCTAA 3)
22
scaffold_5:1-132
(5 CCCTAA 3)
20
scaffold_5:979-1098
3 5
GAP (193 bp)
scaffold_5:785-978
scaffold_5:133-184
GAP (193 bp)
scaffold_5:785-978
scaffold_5:133-184
(5 CCCTAA 3)
20
scaffold_7:1-120
(5 CCCTAA 3)
18
GAP (1680 bp)
scaffold_7:756-2436
3 5
scaffold_7:121-755
(5 CCCTAA 3)
20
scaffold_7:1-120
(5 CCCTAA 3)
18
(5 CCCTAA 3)
20
scaffold_7:1-120
(5 CCCTAA 3)
18
GAP (1680 bp)
scaffold_7:756-2436
3 5
scaffold_7:121-755


402
The screening of the telomere sequences was performed as described above using the
scaffolds of the assemblage v2.0. The basic telomere unit of P. chrysosporium is the heptamer
TTTAGGG. 16 out of the 232 scaffolds contained telomere sequences, eight of them with the
TTTAGGG motif. In four of these scaffolds (numbers 7, 9, 23 y 159) the repetitive unit was
located at an interstitial position at 3200 bp from the 3 end flanked by a gap. This
arrangement could be the result of a wrong assemblage as it can be seen in Fig. 3.

(5 TTTAGGG 3)
22
scaffold_7:2049678-2049832
Terminal sequence (999 bp)
scaffold_7:2050560-2051558
3 5
GAP (726 bp)
scaffold_7:2049833-2050559
Scaffold 9: 1898532 bp (5 TTTAGGG 3)
24
scaffold_9:1896232-1896400
scaffold_9:1896473-1898532
3 5
GAP (71 bp)
scaffold_9:1896401-1896472
(5 TTTAGGG 3)
26
scaffold_23:267927-268109
scaffold_23:272025-273793
3 5
scaffold_23:268344-270904
GAP (1119 bp)
scaffold_23:270905-272024
GAP (233 bp)
scaffold_23:268110-268343
(5 TTTAGGG 3)
16
scaffold_159:3285-3397
scaffold_159:3449-4260
3 5
GAP (50 bp)
scaffold_159:3398-3448
(5 TTTAGGG 3)
22
scaffold_7:2049678-2049832
scaffold_7:2050560-2051558
3 5
GAP (726 bp)
scaffold_7:2049833-2050559
Scaffold 9: 1898532 bp (5 TTTAGGG 3)
24
scaffold_9:1896232-1896400
scaffold_9:1896473-1898532
3 5
GAP (71 bp)
scaffold_9:1896401-1896472
(5 TTTAGGG 3)
26
scaffold_23:267927-268109
scaffold_23:272025-273793
3 5
scaffold_23:268344-270904
GAP (1119 bp)
scaffold_23:270905-272024
GAP (233 bp)
scaffold_23:268110-268343
(5 TTTAGGG 3)
16
scaffold_159:3285-3397
scaffold_159:3449-4260
3 5
GAP (50 bp)
scaffold_159:3398-3448

Fig. 3. Location of telomere regions in scaffolds 5, 9, 23 and 159 of the genome sequence of
P. chrisosporium.


403
Another telomere-like interstitial region was found at about 300000 bp of the 3 end of
scaffold 10. The position of the interstitial telomere block within the scaffold would
suggested that it could be the result of an ancestral intra-chromosomal rearrangement
(inversions and/or fusions), from differential crossing-over or from the repair of double-
strand break during evolution (Lin & Yan, 2008) (Fig. 4).

Fig. 4. Location of telomere regions in scaffold 10 of the genome sequence of
P. chrysosporium.
It was observed that in eight of the 16 telomere-containing scaffolds identified, the repetitive
unit was the complementary CCCTAAA. In five of them, the heptamer unit was placed at
the 5 end. A telomere unit in an internal region at 8772 bp of the 5 end was present in
scaffold 8 while scaffold 5 ferried a telomere motif at 2123492 bp of the 3 end and another
one at 40552bp of the 3 end (Fig. 5).

Fig. 5. Location of telomere regions in scaffolds 5 and 8 of the genome sequence of
P. chrysosporium.
The analysis of scaffold 28 revealed two arrays of interstitial copies of the heptamer
CCCCTAAA (23 and 18 repeats, respectively) placed at 11417 bp and 13853 bp from the 5
(5 TTTAGGG 3)
21
scaffold_10:1195442-1195589
scaffold_10:1195863-1291447
3 5
scaffold_10:1194802-1195441
GAP (273 bp)
scaffold_10:1195590-1195862
GAP (1549 bp)
scaffold_10:1193253-1194801
(5 TTTAGGG 3)
21
scaffold_10:1195442-1195589
scaffold_10:1195863-1291447
3 5
scaffold_10:1194802-1195441
GAP (273 bp)
scaffold_10:1195590-1195862
GAP (1549 bp)
scaffold_10:1193253-1194801
Scaffold 8: 1906386 bp (5 CCCTAAA 3)
33
3 5
(5 TTTAGGG 3)
22
scaffold_5:2123492-2123646
3 5
33
3 5
33
3 5
(5 CCCTAAA 3)
33
3 5
(5 TTTAGGG 3)
22
scaffold_5:2123492-2123646
3 5
(5 TTTAGGG 3)
22
scaffold_5:2123492-2123646
3 5


404
end. An interstitial fragment of 1342 bp and a gap of 933 bp were found between them
suggesting the occurrence of missassemblage (Fig. 6).

Fig. 6. Location of telomere regions in scaffold 28 of the genome sequence of
P. chrysosporium.
In summary, the results presented here suggest that the genome of P. chrysosporium is
arranged in at least eight linkage groups. Because scaffold 8 shows telomere repeats at both
ends it is suggested that the sequence contained in this scaffold constitute the only fully
assembled chromosome of P. chrysosporium.
2.1.3 Analysis of the telomere regions of Pleurotus ostreatus PC15
P. ostreatus PC15 is a monokarytic strain of an industrially-produced edible basidiomycete
that has been also used as a model system for lignocellulose degradation. P. ostreatus differs
from the other white rot model system (P. chrysosporium) in its enzymatic portfolio for lignin
degradation. The structure of its genome was determined by linkage analysis (Larraya et al.,
2000), and the complete genome of this strain has been sequenced. The assemblage v1.0
consists of 19 scaffolds of which 18 were larger than 2 Kbp. The screening of the genome for
telomere sequences was carried out as described above and revealed that the elementary
telomere unit of P. ostreatus is the hexamer TTAGGG. All scaffolds were screened for
telomere regions and 19 telomere regions were recovered. In eight of them the motif
TTAGGG was found, and the remaining 11 had the motif CCCTAA. The number of
repetitions of the basic unit ranged from 19 to 38. As it was determined, scaffolds 1, 3, 4, 5, 6,
9, 10 and 11 show telomere repeats at both ends indicating that they are fully assembled.
2.1.4 Analysis of the telomere regions of Postia placenta MAD-698
P. placenta is a brown rot basidiomycete that rapidly depolymerizes the cellulose in wood
without significant lignin removal. This type of decay differs sharply from white rot fungi
such as P. chrysosporium and P. ostreatus. The genome of the dikaryotic strain of P. placenta
MAD-698 revealed a genome of 90.9 Mbp assembled in 1243 scaffolds (Martinez et al., 2009).
All the scaffolds of the assemblage were screened for telomere sequences as above. The basic
telomere unit in this fungus is the pentamer TTAGG. The analysis of 1243 scaffolds revealed
the presence of 23 regions containing telomere sequences. 12 of them carried the TTAGG
sequence: in eight of them the sequence was found at the scaffolds 3 end, three scaffolds
ferried the sequence in an interstitial location (Fig. 7) and in one of them two regions with
the pentamer sequence appeared at the end of the chromosome but separated by 2373 bp
suggesting a missassemblage of scaffold 178 at its 3 end (Fig. 8).
(5 CCCTAAA 3)
23
scaffold_28:11418-11579
3 5
scaffold_28:11580-12921
(5 CCCTAAA 3)
18
scaffold_28:13855-13981
GAP (933 bp)
scaffold_28:12922-13854
(5 CCCTAAA 3)
23
scaffold_28:11418-11579
3 5
scaffold_28:11580-12921
(5 CCCTAAA 3)
18
scaffold_28:13855-13981
GAP (933 bp)
scaffold_28:12922-13854


405

Fig. 7. Location of telomere regions in scaffolds 37, 117 and 167 of the genome sequence of
P. placenta.

Fig. 8. Location of telomere regions in scaffold 178 of genome sequence of P. placenta.
Scaffold 178: 157039bp
(5 TTAGG 3)
44
scaffold_178:154105-154324
(5 TTAGG 3)
66
scaffold_178:156698-157039
scaffold_178:155854-156697
3 5
GAP (1529 bp)
scaffold_178:154325-155853
(5 TTAGG 3)
44
scaffold_178:154105-154324
(5 TTAGG 3)
66
scaffold_178:156698-157039
scaffold_178:155854-156697
3 5
GAP (1529 bp)
scaffold_178:154325-155853
(5 TTAGG 3)
29
scaffold_37:497534-497679
scaffold_37:497730-501760
3
5
GAP (50 bp)
scaffold_37:497680-497729
scaffold_37:495317-497533
GAP (1391 bp)
scaffold_37:493926-495316
GAP (5892 bp)
scaffold_117:247047-252938
scaffold_117:252939-254534
scaffold_117:245546-247046
(5 TTAGG 3)
50
scaffold_117:245246-245495
GAP (50 bp)
scaffold_117:245496-245545
scaffold_117:244466-245245
GAP (754 bp)
scaffold_117:243712-244465
3 5
(5 TTAGG 3)
31
scaffold_167:165706-
165860<
scaffold_167:165911-167606
3
5
scaffold_167:163789-165705
GAP (50 bp)
scaffold_167:163739-163788
GAP (50 bp)
scaffold_167:165861-165910
(5 TTAGG 3)
29
scaffold_37:497534-497679
scaffold_37:497730-501760
3
5
GAP (50 bp)
scaffold_37:497680-497729
scaffold_37:495317-497533
GAP (1391 bp)
scaffold_37:493926-495316
(5 TTAGG 3)
29
scaffold_37:497534-497679
scaffold_37:497730-501760
3
5
GAP (50 bp)
scaffold_37:497680-497729
scaffold_37:495317-497533
GAP (1391 bp)
scaffold_37:493926-495316
GAP (5892 bp)
scaffold_117:247047-252938
scaffold_117:252939-254534
scaffold_117:245546-247046
(5 TTAGG 3)
50
scaffold_117:245246-245495
GAP (50 bp)
scaffold_117:245496-245545
scaffold_117:244466-245245
GAP (754 bp)
scaffold_117:243712-244465
3 5
GAP (5892 bp)
scaffold_117:247047-252938
scaffold_117:252939-254534
scaffold_117:245546-247046
(5 TTAGG 3)
50
scaffold_117:245246-245495
GAP (50 bp)
scaffold_117:245496-245545
scaffold_117:244466-245245
GAP (754 bp)
scaffold_117:243712-244465
3 5 3 5
(5 TTAGG 3)
31
165860<
scaffold_167:165911-167606
3
5
scaffold_167:163789-165705
GAP (50 bp)
scaffold_167:163739-163788
GAP (50 bp)
scaffold_167:165861-165910
(5 TTAGG 3)
31
165860<
scaffold_167:165911-167606
3
5
scaffold_167:163789-165705
GAP (50 bp)
scaffold_167:163739-163788
GAP (50 bp)
scaffold_167:165861-165910


406
The remaining 11 scaffolds carried the telomere unit CCTAA placed at the 5end in seven
of them. The scaffold 99 (Fig. 9) has a complex structure with two interstitial CCTAA
regions containing the motif. One of them located towards the scaffold 5 end, contains 40
copies of the telomere unit, and the other, located 16533 bp downstream, another
interstitial region containing 19 repetitions of the unit. A gap of about 50 bp placed at the
5end of this interstitial region suggests that this arrangement could be due to a
missassemblage.

The scaffold 33 also showed two interstitial regions with 40 and 30 repetitions of the of the
CCTAA telomere motif. One of the regions was preceded by a gap of 701 bp suggesting that
it could be a wrong assemblage. The other one (40 copies of the telomere unit) could very
well represent and ITS (Interstitial Telomere Sequence) that can be produced by
chromosome rearrangements as described above.

Two other scaffolds with interstitial sequences were found. Scaffold 70 showed 26
repetitions of the CCTAA telomere unit at 38395 bp from the 5 end preceded by a gap, and
scaffold 144 showed 50 repetitions of the telomere unit at 181041 bp from the 5 end
preceded by another other gap of 50 bp (Fig. 11). We suggest that these structures are
consequence of wrong assemblages.
3 5
(5 CCTAA 3)
40
scaffold_99:
1-202
(5 CCTAA 3)
19
scaffold_99:
16756-16850
GAP (2309 bp)
scaffold_99:
1217-3525
GAP (1118 bp)
scaffold_99:
8003-9120
GAP (1848 bp)
scaffold_99:
10098-11945
GAP (1053 bp)
scaffold_99:
13319-14371
GAP (50 bp)
scaffold_99:
15515-15564
GAP (50 bp)
scaffold_99:
16706-16755
scaffold_99:
203-1216
scaffold_99:
3526-8002
scaffold_99:
9121-10097
scaffold_99:
11946-13318
scaffold_99:
14372-15514
scaffold_99:
15565-16705
3 5
(5 CCTAA 3)
40
scaffold_99:
1-202
(5 CCTAA 3)
19
scaffold_99:
16756-16850
GAP (2309 bp)
scaffold_99:
1217-3525
GAP (1118 bp)
scaffold_99:
8003-9120
GAP (1848 bp)
scaffold_99:
10098-11945
GAP (1053 bp)
scaffold_99:
13319-14371
GAP (50 bp)
scaffold_99:
15515-15564
GAP (50 bp)
scaffold_99:
16706-16755
(5 CCTAA 3)
40
scaffold_99:
1-202
(5 CCTAA 3)
19
scaffold_99:
16756-16850
GAP (2309 bp)
scaffold_99:
1217-3525
GAP (1118 bp)
scaffold_99:
8003-9120
GAP (1848 bp)
scaffold_99:
10098-11945
GAP (1053 bp)
scaffold_99:
13319-14371
GAP (50 bp)
scaffold_99:
15515-15564
GAP (50 bp)
scaffold_99:
16706-16755
scaffold_99:
203-1216
scaffold_99:
3526-8002
scaffold_99:
9121-10097
scaffold_99:
11946-13318
scaffold_99:
14372-15514
scaffold_99:
15565-16705
scaffold_99:
203-1216
scaffold_99:
3526-8002
scaffold_99:
9121-10097
scaffold_99:
11946-13318
scaffold_99:
14372-15514
scaffold_99:
15565-16705
(5 CCTAA 3)
40
scaffold_33:279914-280114
(5 CCTAA 3)
30
scaffold_33:281476-281625
GAP (701 bp)
scaffold_33:280775-281475
3 5
scaffold_33:280115-280774
(5 CCTAA 3)
40
scaffold_33:279914-280114
(5 CCTAA 3)
30
scaffold_33:281476-281625
GAP (701 bp)
scaffold_33:280775-281475
3 5
scaffold_33:280115-280774


407

Fig. 11. Location of telomere regions in scaffolds 70 and 144 of genome sequence of
P. placenta.
In summary, P. placenta has a telomere pentameric (TTAGG) basic repetitive unit. The
number of copies present in the assembled genome ranged from 19 to 70. The analysis of
data suggests that the minimum number of linkage groups of this species could be 12.
2.1.5 Analysis of the telomere regions of Heterobasidion annosum
Heterobasidion annosum is a root pathogen responsible for important losses in conifer
plantations and natural forests throughout the northern hemisphere (Asiegbu et al., 2005).
Genetic linkage analyses of this fungus had produced maps with 19 large linkage groups and
20 smaller ones (Lind et al., 2005), but the precise chromosome number for this species has not
been conclusively determined. The v2.0 of the homokaryotic H. annosum genome assembly
consists of 33.1 Mbp sequence assembled into 15 scaffolds at least 10 of which represent nearly
complete chromosomes (http://genome.jgipsf.org/Hetan2/Hetan2.home.html).
The screening of the telomere sequences was performed as it was described above. The H.
annosum telomere repetitive sequence is a TTAGG pentamer. The screening of the 15
scaffolds rendered 19 telomere regions. Six of them corresponded to the direct repeat
sequence at the 3 end of the scaffolds, and the remaining 13 carried the reverse sequence
CCTAA at the scaffolds 5 end. These results suggest that the genome of H. annosum is
arranged in at least 13 linkage groups. Taking into account that scaffolds 5, 6, 9, 10, 11 and
12 contained telomere repeats at both ends, it can be concluded that they could correspond
to fully assembled chromosomes.
2.1.6 Summary of the telomere regions of different basidiomycetes
A summary of the structural characteristics of the telomeres studied in this paper can be
found in Table 1.
3 5
scaffold_70:38531-39144
GAP (1081 bp)
scaffold_70:36237-37317
GAP (50 bp)
scaffold_70:38346-38395
(5 CCTAA 3)
26
scaffold_70:38396-38530
scaffold_70:37318-38345
GAP (1080 bp)
scaffold_70:39145-40224
3 5
GAP (493 bp)
scaffold_144:176574-177067
GAP (50 bp)
scaffold_144:179227-179276
(5 CCTAA 3)
50
scaffold_144:181042-181291
scaffold_144:181292-181991
scaffold_144:179277-180991
scaffold_144:177068-179226
GAP (50 bp)
scaffold_144:180992-181041
GAP (380 bp)
scaffold_144:181992-182371
3 5
scaffold_70:38531-39144
GAP (1081 bp)
scaffold_70:36237-37317
GAP (50 bp)
scaffold_70:38346-38395
(5 CCTAA 3)
26
scaffold_70:38396-38530
scaffold_70:37318-38345
GAP (1080 bp)
scaffold_70:39145-40224
3 5
scaffold_70:38531-39144
GAP (1081 bp)
scaffold_70:36237-37317
GAP (50 bp)
scaffold_70:38346-38395
scaffold_70:38531-39144
GAP (1081 bp)
scaffold_70:36237-37317
GAP (50 bp)
scaffold_70:38346-38395
(5 CCTAA 3)
26
scaffold_70:38396-38530
scaffold_70:37318-38345
GAP (1080 bp)
scaffold_70:39145-40224
(5 CCTAA 3)
26
scaffold_70:38396-38530
scaffold_70:37318-38345
GAP (1080 bp)
scaffold_70:39145-40224
3 5
GAP (493 bp)
scaffold_144:176574-177067
GAP (50 bp)
scaffold_144:179227-179276
(5 CCTAA 3)
50
scaffold_144:181042-181291
scaffold_144:181292-181991
scaffold_144:179277-180991
scaffold_144:177068-179226
GAP (50 bp)
scaffold_144:180992-181041
GAP (380 bp)
scaffold_144:181992-182371
3 5
GAP (493 bp)
scaffold_144:176574-177067
GAP (50 bp)
scaffold_144:179227-179276
(5 CCTAA 3)
50
scaffold_144:181042-181291
scaffold_144:181292-181991
GAP (493 bp)
scaffold_144:176574-177067
GAP (50 bp)
scaffold_144:179227-179276
(5 CCTAA 3)
50
scaffold_144:181042-181291
scaffold_144:181292-181991
scaffold_144:179277-180991
scaffold_144:177068-179226
GAP (50 bp)
scaffold_144:180992-181041
GAP (380 bp)
scaffold_144:181992-182371
scaffold_144:179277-180991
scaffold_144:177068-179226
GAP (50 bp)
scaffold_144:180992-181041
GAP (380 bp)
scaffold_144:181992-182371


408
Species
Genome
length
assembled
Scaffold
number
Telomere
repetition
Average
number of
telomere
Minimum number
of linkage groups
analyzed
C. subvermispora 39.0 Mb 740 TTAGGG 19 copies 11
P. chrysosporium 35.1 Mb 232 TTTAGGG 22 copies 8
P. ostreatus 34.3 Mb 12 TTAGGG 24 copies 11
P. placenta 90.0 Mb 1243 TTAGG 25 copies 12
H. annosum 33.1 Mb 15 TTAGG 25 copies 13
Table 1. Structural characteristics of the telomeres in the basidiomycetes analyzed.
2.2 Analysis of the basidiomycetes subtelomeric regions
The analysis of the subtelomeric regions is aimed at answering two questions: are the genes
sitting at the subtelomeric a representative sample of the genes of each species or is there
enrichment in sub-telomere specific genes? If this were the case, which are these telomere-
enriched genes and are they conserved across species? In order to address these questions,
we have recorded the genes automatically annotated in 50 Kbp regions adjacent to the
different telomeres identified in the species analyzed, we have checked them manually and
we have recorded and classified the Gene Ontology (GO) terms related to the genes
identified in these regions (Ashburner et al., 2000).
As the number of telomere-containing scaffolds differed in the various species, (from 12 in P
ostreatus to 1243 in P. placenta) the length of genomic sequence screened also varied,
although in a much smaller degree (from 800 Kbp in P. chrysosporium to 2100 Kbp in C.
subvermispora). The gene density of the analyzed regions was found to be related to the
degree of finishing of the genome: those assembled as draft (C. subvermispora, P. placenta and
P. chrysosporium) have gene densities lower than 0.20 genes per Kbp, whereas the gene
density in the finished genomes is much higher (0.34 and 0.49 genes per Kbp). The gene
density in the draft genomes seems to be significantly lower than the global gene density in
the corresponding genomes. This can be due to deficiencies in the annotation of these draft
genomes since the global gene density in all the species analyzed (with the exception of P.
placenta) is very similar. In all cases, the most of the genes automatically annotated at the
subtelomeric regions had no homology with others of the gene databases (cutoff criterion e-
value<e-20 for BlastX) (Table 2).
The Gene Ontology annotation is aimed at standardizing the representation of gene and
gene products in such a way that they can be compared among databases. This approach
project provides a controlled vocabulary of terms for describing gene product characteristics
and gene product annotation data (Ashburner et al., 2000). There are three classification
categories that are provided by the consortium: Biological Process (BP), Cellular Component
(CC) and Molecular Function (MF). Each identified gene product is labeled with all the GO
terms in each category that can define it. By this way, a list of GO terms provides a kind of
picture describing the specific condition of the gene subset that is under study. We have
studied the three categories of GO annotation in the genes annotated in 50 Kbp subtelomeric
regions in the five genomes studied and recorded their general statistics (Table 3). Because
of the low number of gene models identified in the subtelomeric regions and because not all
of them can be labeled with a GO term, the numbers of terms in the categories of Biological


409
Feature C.
subvermispora
P.
chrysosporium
P. ostreatus P. placenta H. annosum
Total number of scaffolds 740 232 12 1243 15
Number of subtelomeric
regions analyzed
42 16 19 23 19
Scaffolds with direct motif 22 8 8 12 6
Scaffolds with reverse motif 20 8 11 11 13
Total length analyzed (Kbp) 2100 800 950 1150 950
Total number filtered model
genes
283 134 319 177 470
Unknown genes
(no homology)
134 (47.3 %) 56 (41.8 %) 87 (27.3 %) 64 (36.2 %) 279 (59.4 %)
Predicted protein 104 (36.7 %) 57 (42.5 %) 182 (57.1 %) 79 (44.6 %) 131 (27.9 %)
Known/annotated/putative
genes
45 (15.9 %) 21 (15.7 %) 50 (15.7 %) 34 (19.2 %) 60 (12.8 %)
Subtelomere gene density
(genes / Kbp)
0.13 0.17 0.34 0.10 0.49
Whole genome gene density
(genes / Kbp)
0.31 0.39 0.34 0.19 0.40
Table 2. Density and homology types in the subtelomere regions.
Processes and Cellular Component are rather low, whereas the number of Molecular
Function terms is much higher and produces a clearer picture of what are the subtelomeric
regions coding for (Table 3).

C.
subvermispora
P.
chrysosporium
P.
ostreatus
P.
placenta
H.
annosum
GO category
Biological Process (BP) 46 22 24 20 48
Cellular Component (CC) 15 9 10 7 12
Molecular Function (MF) 127 88 161 65 186
Table 3. GO terms richness in the subtelomeric regions of the five basidiomycetes analyzed.
In order to determine if the genes found at the subtelomere constitute a representative
sample of the genes of each species, we can perform a simple statistical analysis to calculate
the numbers that would be expected for each one of the GO terms in the subtelomeric
regions using the whole genome data as frequency. If we do this type of study, we conclude
that the distribution of the subtelomeric GO terms for each one of the categories is not a
representative sample of the total gene set for each one of the species (data not shown) and,
consequently, we can conclude that there are sets of genes that are found more frequently at
the telomere regions. For identifying these sets, we must discuss the GO term distribution in
each one of the species.
2.2.1 Analysis of the subtelomere regions of Ceriporiopsis subvermispora B
The analysis of the 283 gene models annotated in the subtelomeric regions of C.
subvermispora revealed 46 BP terms in which transport, protein amino acid phosphorylation,


410
metabolic process, electron transport, carbohydrate metabolic process, and proteolysis are
the more represented ones. The terms transport, protein amino acid phosphorylation and
carbohydrate metabolism seem to be overrepresented in this region whereas the terms
metabolic processes and electron transport seem to be underrepresented in comparison with
the total genome. The analysis revealed 15 CC terms annotated in this region. Out of which,
the terms intracellular, integral to membrane, membrane and nucleus are the most
represented ones in this category. All of them, except the term nucleus, seem to be
overrepresented in the subtelomeric region. Finally, out of the 127 MF terms being the more
represented were ATP binding, zinc ion binding, nucleic acid binding protein, binding
protein, kinase activity, protein serine/threonine kinase activity, transporter activity,
oxidoreductase activity, and protein-tyrosine kinase activity. All of them seem to be
overrepresented in the subtelomeric regions in comparison to the whole genome.
2.2.2 Analysis of the subtelomere regions of Phanerochaete chrysosporium
The analysis of the 134 gene models annotated in the subtelomeric regions of P.
chrysosporium revealed 22 BP terms being the most represented terms proteolysis and
peptidolysis, electron transport, metabolism, methionine biosynthesis, protein transport,
small GTPase mediated signal transduction, and transport. The analysis revealed 9 CC
terms out of which, the terms membrane, nucleus and integral to membrane are the most
represented ones in this category. Finally, out of the 88 MF terms, the more represented
terms are those of aspartic-type endopeptidase activity, nucleic acid binding, zinc ion
binding, ATP binding, and oxidoreductase activity
2.2.3 Analysis of the subtelomere regions of Pleurotus ostreatus
The analysis of the 319 gene models annotated in the subtelomeric regions of P. ostreatus
revealed 24 BP terms being the most represented terms protein amino acid phosphorylation,
proteolysis, metabolic process, electron transport, and transport. The analysis revealed 10
CC terms out of which, the terms intracellular, integral to membrane, nucleus, cell wall and
ribosome are the most represented ones in this category. Finally, out of the 161 MF terms,
the more represented terms are those of ATP binding, nucleic acid binding, oxidoreductase
activity, protein-tyrosine kinase activity and zinc ion binding.
2.2.4 Analysis of the subtelomere regions of Postia placenta
The analysis of the 177 gene models annotated in the subtelomeric regions of P. placenta
revealed 20 BP terms being the most represented terms proteolysis, electron transport,
protein amino acid phosphorylation and regulation of transcription DNA-dependent. The
analysis revealed 7 CC terms out of which, the terms intracellular, membrane, and nucleus
are the most represented ones in this category. Finally, out of the 65 MF terms, the more
represented terms are those of ATP binding, nucleic acid binding and zinc ion binding.
2.2.5 Analysis of the subtelomere regions of Heterobasidion annosum
The analysis of the 470 gene models annotated in the subtelomeric regions of H. annosum
revealed 48 BP terms being the most represented terms metabolic process, transport,
electron transport, regulation of transcription DNA-dependent, carbohydrate metabolic
process, and proteolysis. The analysis revealed 12 CC terms out of which, the terms
membrane, integral to membrane, intracellular, nucleus and cytoplasm are the most


411
represented ones in this category. Finally, out of the 186 MF terms, the more represented
terms are those of zinc ion binding, oxidoreductase activity, ATP binding, binding and heme
binding.
2.2.6 Comparative analysis of the subtelomere regions of the five basidiomycetes
The record of the GO terms associated to genes found in the 50 Kbp adjacent to the telomere
sequences in the five basidiomycetes analyzed reveals common patterns that permit to
determine some telomere-enriched GO term families. As a preliminary study, we have taken
into account the terms that are always present in among the most represented ones in each
of the categories and we have extracted those of them that are present in all or most of the
species analyzed. If we consider the BP category, the term electron transport is among the
most represented in the five species studied and the terms transport, protein aminoacid
phosphorylation, metabolic process and proteolysis are present in four of the five species.
So, we can conclude that the subtelomeric regions are enriched in these processes. The
species C. subvermispora, H. annosum, and P. ostreatus have subtelomeric regions where the
more abundant BP-GO terms are highly similar whereas the subtelomeric regions in P.
chrysosporium are the most dissimilar.
If we consider the CC category, the terms nucleus and intracellular are present among the
more represented ones in all the species studied, the terms membrane and integral to
membrane are present in four of the species and the terms ribosome and cytoplasm are
present in three of the five species. In this case the more different subtelomeric regions in
terms of the CC-GO terms are those of C. subvermispora and P. placenta, being the other three
species in intermediate positions.
Finally, in the case of the MF terms, as their number is much higher, a deeper comparison
can be made among the species (Table 4). The five species analyzed share the most frequent
MF terms associated to the genes in the subtelomere regions supporting the idea of a
preference for certain gene of gene families at these chromosome locations.

Molecular function
Ceriporiopsis
subvermispora
Phanerochaete
chrysosporium
Pleurotus
ostreatus
Postia
placenta
Heterobasidion
annosum
Presence
zinc ion binding 4,76 3,60 2,02 4,96 4,72 5
oxidoreductase
activity
2,60 2,70 2,69 2,48 3,14 5
ATP binding 5,63 2,70 5,05 6,61 2,83 5
nucleic acid binding 3,90 4,50 4,38 4,96 2,20 5
catalytic activity 2,16 1,80 1,35 2,48 2,20 5
DNA binding 1,30 0,90 1,68 2,48 1,57 5
transporter activity 3,03 1,80 1,35 ND 2,20 4
iron ion binding 1,35 1,65 1,57 3
protein-tyrosine
kinase activity
2,60 2,02 2,48 3
Table 4. Frequency of Molecular Function GO terms in the subtelomere regions of the
studied basidiomycetes.


412
3. Synteny
Synteny can be defined as the conservation of the relative positions and order of genes in
different chromosomes. This definition implies that the conserved genes are related by their
descent from an original ancestor (homologous genes). There are two types of homology:
orthology and paralogy. We can call two genes belonging to different species as orthologous
if they descent from a single gene present in the last common of the two species. On the
other hand, two genes are called paralogous, if they derive from gene duplication events
occurred in a given species. The orthology requires that speciation has occurred, whereas
this is not necessary in the case of parology, which can occur only in individuals of the same
species. As the evolutionary histories of different species may differ, groups of paralogous
genes can be orthologous of a single gene in a different species. The preserved co-
localization of genomic regions on chromosomes of different species is called shared
synteny. This may involve relationships between genes within the syntenic regions
involved, such as combinations of alleles that are advantegeous when inherited together, or
shared regulatory mechanisms.
The problem of identifying syntenic regions in different genomes has been addressed using
different strategies including the use of FASTA (Lipman & Pearson, 1985) and Blast
(Altschul et al., 1997), and different bioinformatics approaches (Catchen et al., 2009;
Grabherr et al., 2010; Tang et al., 2011). We have used a method based on the identification
of synteny regions at the chromosome ends by means of a BLASTP analysis of genes in the
two genomes using a cut-off threshold of e-20. Later, the Vista Synteny Viewer
(http://genome.lbl.gov/vista/index.shtml) integrated into the JGI Genome Portal was used
in the preliminary orthologous searching of each species. This tool enables pair-wise
comparative analysis of genome assemblies at three levels of resolution. The use of synteny
software programs is of particular interest to see the particular changes undergone by the
subtelomeric regions during evolution (Housworth & Postlethwait, 2002). For instance, the
chromosome 3 in H. annosum maintains the synteny with P. ostreatus chromosome 3, but the
H. annosum subtelomeric region aligned with a central region of P. ostreatus chromosome 4
suggesting the occurrence of a translocation event after the divergence of the two species.
The different basidiomycetes were used as reference genomes and P. ostreatus PC15 v2.0 was
the query genome. Focusing in the distal 50Kbp of each chromosome, we identified the
putative gene orthologous in the subtelomeric regions of each basidiomycete. Then we used
that gene sequences as query in a BlastP search of the P. ostreatus filtered model genes as
subject. Two models were considered as orthologous if their alignment had an e-value lower
than e-20 and they shared a minimum 60% in identity percentage.
The synteny between the subtelomeric regions was analyzed using P. ostreatus chromosomes
as a reference. It was observed that seven P. ostreatus chromosomes (chromosomes 1, 3, 4, 5,
7, 8 and 10) harbored sequences homologous to subtelomere regions of the other
basidiomycetes analyzed in this study (Table 5). 47 synteny regions were uncovered when
the subtelomeric regions of C. subvermispora were compared to those of P. ostreatus. The
highest number (16) corresponded to those regions placed at P. ostreatus chromosome 7.
However, it should be mentioned that 12 C. subvermispora gene models were found within a
30 Kbp region of the P. ostreatus genome (data not shown). The lowest number of synteny
regions was found when the subtelomeric regions of P. placenta were used as query. It


413
should be noticed that 10 out of 15 regions were placed on chromosome 5 of P. ostreatus. A
similar situation was observed when synteny was analyzed between H. annosum and P.
ostreatus. 10 out of 25 synteny regions of H. annosum mapped to chromosome 10 of P.
ostreatus.

P. ostreatus chromosome
Chr 1 Chr 3 Chr 4 Chr 5 Chr 7 Chr 8 Chr 10 Total
C. subvermispora 6 6 7 16 12 47
P. chrysosporium 4 4 4 12
P. placenta 5 10 15
H. annosum 6 9 10 25
Total 10 6 22 19 20 12 10 101

Table 5. Number of subtelomeric synteny regions in different basidiomycetes using
P. ostreatus as reference.
The chromosome 4 of P. ostreatus can be defined as mosaic of modules of subtelomeres from
the other basidiomycetes studied in this paper. The list below contains some gene models
mapping to the subtelomeric regions in these basidiomycetes that were found at interstitial
positions in P. ostreatus chromosome 4: from C. subvermispora, a cell cycle check point
protein, a membrane transporter, a histone deacetylase, a histidine acid phosphatase, the
ribosomal protein L1 and an ABC transporter; from P. chrysosporium, a haloacid
dehalogenase-like hydrolase and a glycoside hydrolase; from P. placenta, an inositol
polyphosphate phosphatase, a metal-dependent phosphohydrolase, and a monooxygenase;
from H. annosum, a mitochondrial carrier transporter, a Golgi transporter, and zinc finger
transcription factor (Fig. 12).
11 out of 12 gene models of C. subvermispora were syntenic to a 20 Kbp regions of P. ostreatus
chromosome 8. These gene models corresponded to a nucleic acid binding protein, citrate
synthase, a methyltransferase, an exoribonuclease, a phosphoribosyltransferase, a
prenyltransferase, a homeobox transcription factor, a mitochondrial inner membrane
protein importer, as DNA-J type heat shock protein, a RNA splicing protein, and a
cytochrome c oxidase.
The genome of P. falciparum is organized in 14 compartmentalized chromosomes where the
conserved regions form the central chromosomal domains and the polymorphic regions are
at the terminal domains. In this way, housekeeping genes tend to be located at the central
regions of the chromosomes, whereas the highly variable gene families responsible for the
antigenic variation of the parasite are clustered towards the telomeres (Hernandez-Rivas et
al., 2010). Our results suggest that a similar type of chromosomal organization would be
expected to occur in basidiomycetes, although a larger number of genomes should be
studied to fully support this hypothesis.


414
Ppla_42906
Post_50083
Post_1040099
Post_1111873
(CCCTAA)
38
Post_1096328
Post_1029741
Post_27447
Post_1021683
Post_1063555
Post_1102815
410,000
415,000
1
10,000
20,000
255,000
260,000
1,120,000
1,130,000
662,000
672,000
682,000
692,000
702,000
712,000
722,000
Chromosome
position
Pchr_129484
Pchr_2004
Pchr_35714
Pchr_2003
Ppla_46863
Ppla_93579
Ppla_93580
Ppla_46929
3,275,000
3,280,000
3,425,000
3,430,000
3,335,000
3,345,000
3,385,000
3,390,000
3,480,000
3,500,000
3,510,000
3,490,000
3,515,000
2,965,000
2,975,000
2,985,000
1,295,000
1,305,000
Post_26215
Post_1103034
Csub_148142
Csub_110873
Csub_110874
Csub_110877
Csub_110878
Csub_79948
Csub_45036
Post_157567
Post_39620
Post_26234
Post_26915
Post_185946
Post_176197
Hann_17090
Hann_47641
Hann_314581
Hann_432754
Hann_313250
Hann_48385
Post_1093096
Post_1064481
Post_1064483
Post_1063733
Post_1015429
TTAGGG
Ppla_42906
Post_50083
Post_1040099
Post_1111873
(CCCTAA)
38
Post_1096328
Post_1029741
Post_27447
Post_1021683
Post_1063555
Post_1102815
410,000
415,000
1
10,000
20,000
255,000
260,000
1,120,000
1,130,000
662,000
672,000
682,000
692,000
702,000
712,000
722,000
Chromosome
position
Pchr_129484
Pchr_2004
Pchr_35714
Pchr_2003
Ppla_46863
Ppla_93579
Ppla_93580
Ppla_46929
3,275,000
3,280,000
3,425,000
3,430,000
3,335,000
3,345,000
3,385,000
3,390,000
3,480,000
3,500,000
3,510,000
3,490,000
3,515,000
2,965,000
2,975,000
2,985,000
1,295,000
1,305,000
Post_26215
Post_1103034
Csub_148142
Csub_110873
Csub_110874
Csub_110877
Csub_110878
Csub_79948
Csub_45036
Post_157567
Post_39620
Post_26234
Post_26915
Post_185946
Post_176197
Hann_17090
Hann_47641
Hann_314581
Hann_432754
Hann_313250
Hann_48385
Post_1093096
Post_1064481
Post_1064483
Post_1063733
Post_1015429
TTAGGG
Ppla_42906
Post_50083
Post_1040099
Post_1111873
(CCCTAA)
38
Post_1096328
Post_1029741
Post_27447
Post_1021683
Post_1063555
Post_1102815
410,000
415,000
1
10,000
20,000
255,000
260,000
1,120,000
1,130,000
662,000
672,000
682,000
692,000
702,000
712,000
722,000
Chromosome
position
Pchr_129484
Pchr_2004
Pchr_35714
Pchr_2003
Ppla_46863
Ppla_93579
Ppla_93580
Ppla_46929
Ppla_42906
Post_50083
Post_1040099
Post_1111873
(CCCTAA)
38
Post_1096328
Post_1029741
Post_27447
Post_1021683
Post_1063555
Post_1102815
Post_50083
Post_1040099
Post_1111873
(CCCTAA)
38
Post_1096328
Post_1029741
Post_27447
Post_1021683
Post_1063555
Post_1102815
410,000
415,000
1
10,000
20,000
255,000
260,000
1,120,000
1,130,000
662,000
672,000
682,000
692,000
702,000
712,000
722,000
Chromosome
position
Pchr_129484
Pchr_2004
Pchr_35714
Pchr_2003
Ppla_46863
Ppla_93579
Ppla_93580
Ppla_46929
3,275,000
3,280,000
3,425,000
3,430,000
3,335,000
3,345,000
3,385,000
3,390,000
3,480,000
3,500,000
3,510,000
3,490,000
3,515,000
2,965,000
2,975,000
2,985,000
1,295,000
1,305,000
Post_26215
Post_1103034
Csub_148142
Csub_110873
Csub_110874
Csub_110877
Csub_110878
Csub_79948
Csub_45036
Post_157567
Post_39620
Post_26234
Post_26915
Post_185946
Post_176197
Hann_17090
Hann_47641
Hann_314581
Hann_432754
Hann_313250
Hann_48385
Post_1093096
Post_1064481
Post_1064483
Post_1063733
Post_1015429
TTAGGG
3,275,000
3,280,000
3,425,000
3,430,000
3,335,000
3,345,000
3,385,000
3,390,000
3,480,000
3,500,000
3,510,000
3,490,000
3,515,000
2,965,000
2,975,000
2,985,000
1,295,000
1,305,000
Post_26215
Post_1103034
Csub_148142
Csub_110873
Csub_110874
Csub_110877
Csub_110878
Csub_79948
Csub_45036
Post_157567
Post_39620
Post_26234
Post_26915
Post_185946
Post_176197
Hann_17090
Hann_47641
Hann_314581
Hann_432754
Hann_313250
Hann_48385
Post_1093096
Post_1064481
Post_1064483
Post_1063733
Post_1015429
Post_26215
Post_1103034
Csub_148142
Csub_110873
Csub_110874
Csub_110877
Csub_110878
Csub_79948
Csub_45036
Post_157567
Post_39620
Post_26234
Post_26915
Post_185946
Post_176197
Hann_17090
Hann_47641
Hann_314581
Hann_432754
Hann_313250
Hann_48385
Post_1093096
Post_1064481
Post_1064483
Post_1063733
Post_1015429
TTAGGG

Fig. 12. Mosaic structure of P. ostreatus chromosome 4. The syntenic gene models of P.
ostreatus (Post), P. chrysosporium (Pchr), P. placenta (Ppla) and H. annosum (Hann) are
indicated along with their position on the P. ostreatus chromosome.


415
4. Conclusion
The bioinformatics analysis described in this paper allowed us to establish the type and the
number of the telomere repeat unit in the basidiomycetes analyzed, to suggest the putative
linkage groups in fungi where linkage maps are not available, to uncover misassembled
telomere regions, and to reveal the preference for some gene models to be located at the
subtelomeric regions and to uncover synteny among the subtelomere regions in the
basidiomycetes analyzed.
5. Acknowledgements
This work has been supported by funds of the AGL2008-05608-C02-01 of the Spanish
National Plan of Scientific Research, the Bioethanol Euroinnova project of the Goverment of
Navarre (Spain), by additional institutional support from the Public University of Navarre.
Some of the sequence data were produced in genome sequence projects developed at the JGI
within the Community Sequence Program under the auspices of the US Department of
Energy's Office of Science, Biological and Environmental Research Program, and by its
associate National Laboratories Lawrence Livermore and Los Alamos.
LR led and coordinated the project, GP determined bioinformatically the telomeres and
subtelomere regions in the species. RC, FS and AGP made the GO analysis of the data. The
manuscript was prepared by LR, and AGP.
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20
SNPpattern: A Genetic Tool to Derive
Haplotype Blocks and Measure Genomic
Diversity in Populations Using SNP Genotypes
Stephen J. Goodswen
1,2
and Haja N. Kadarmideen
3

1
University of Technology Sydney, Broadway, Sydney, NSW
2
CSIRO Livestock Industries, ATSIP, University Drive,
James Cook University Campus, Townsville, QLD
3
Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences,
University of Copenhagen, Frederiksberg C
1,2
Australia
3
Denmark
1. Introduction
The aftermath of the Human Genome Project has generated new revolutionary techniques
and equipment such as high throughput measurement tools for collecting biological
information. One notable tool is a microarray that can be used to genotype hundreds of
thousands of single nucleotide polymorphisms (SNPs) in one run. This highthroughput SNP
genotypes along with phenotypic measurements can be used in fine quantitative trait loci
(QTL) mapping or genome-wide association studies (GWAS). The result of fine QTL
mapping or GWAS is a set of statistically significant QTL regions or genetic markers such as
SNPs. See Box 1 for SNP, QTL and GWAS explanation. The significant QTLs or SNPs from
QTL mapping or GWAS are used subsequently in QTL or SNP based selection of elite
animals or plants for breeding in agriculture or used to predict disease risks in humans and
animals (e.g. Burton et al. 2007, Mackay et al. 2009). GWAS relies on a natural phenomenon
of linkage disequilibrium (LD) between genetic (SNP) markers and causal variants or
quantitative trait nucleotide (QTN). For GWAS to be applied successfully there is a need to
understand the extent and distribution of linkage disequilibrium (LD) across the entire
genome in a population. In particular, we need to know how LD varies from one region (or
population) to another. This need to know how LD (and haplotype diversity) varies from
one region or population to another provided the motivation to develop SNPpattern, a
generic bioinformatic tool for finding SNP allele patterns in populations.
1.1 The principles of linkage disequilibrium (LD) and haplotypes
We are currently in a bioinformatics era. The emergence of bioinformatics is the result of
two converging forces. One relates to the exponential increase in computer processing
power, digital storage capacity, and digital communication. The other force is the
exponential increase in biological data (Larranaga et al., 2006). Prior to the 1990s biologists

could be stereotyped as being isolated in their experimental laboratories doing their poorly
funded projects and recording their findings in a paper format. The Human Genome Project
completely changed all of this (Collins et al., 2003). Notwithstanding the staggering $3
billion cost for the project, the scientific findings and the new revolutionary techniques and
equipment have spurred on many other projects to generate an avalanche of advances in
gene technologies, genomics, and molecular biology. Some of the notable developments are
the high throughput measurement tools for collecting biological information; tools such as
microarrays, high speed DNA sequencers, and mass spectrometers. The main outcome from
all this new technology is enormous amounts of disseminated biological data in different
digital formats. One of the main challenges in bioinformatics is to transform the
exponentially growing biological data into useful information. What constitutes useful
information is of course debatable; nevertheless, information is the critical starting
component to solving biological problems. Living cells are extremely complicated systems,
even so, the new high throughput measurement tools have revolutionised the way we can
collect biological data about these systems and begin to unravel the complexity. In the light
of these advances in genomics, the bioinformatics aspiration is to provide the relevant tools
to make sense of multiple sources of omics datasets or at the very least, enable the
researcher to make valuable inferences, connections and predictions from the information.
Kadarmideen & Reverter (2007) provided a good review of some integrative analytical
framework combining multiple -omics data types specifically for livestock populations but
they discuss generic issues for most species where genome sequences are being made
available. For instance, Kadarmideen et al., (2006), Kadarmideen and Janss (2007) and
Kadarmideen (2008), apply an integrative systems genetics approaches to map genetic
variants and unravel underlying genetic networks of diabetes, stress, and reproduction,
respectively in recombinant inbred strains of mouse genotyped for over 2 million SNP
genetic markers and microarray expression profiled for over 20000 transcripts in various
tissues. Without the relevant bioinformatics tools, it would not have been possible to
integrate such large datasets and apply sophisticated statistical genetic algorithms and
models.
Systematic studies of common genetic variants have shown that some combinations of
polymorphisms at different loci occur more or less frequently in a population such that the
alleles of these polymorphisms are associated more often than if they were unlinked. That is,
there is a statistically significant difference between observed and expected allelic
frequencies (expected, in this instance, refers to allelic frequencies as result of independent
segregation).
This non-random and non-Mendelian association between alleles at two or more loci is
referred to as linkage disequilibrium (LD) and is a departure from the Hardy-Weinberg
equilibrium. SNPs (Box 1) are the most common polymorphism and are extremely dense
throughout the genome which allows for an effective study of common haplotypes. For the
remaining of this section, SNPs will be used when referring to variants/polymorphism in
the context of LD.
Prior to the year 2004 there was little published research on LD in humans, yet from 2004
onwards an exponential release of publications commenced
1
(for instance, see patterns of
human LD in Ardlie et al. 2002). It is argued that this increase in interest is mainly because
of the increased applications of LD as a tool. For example, LD is the essential tool of genetic

1
Based on ISI Web of Knowledge
SM
searches
SNPpattern: A Genetic Tool to Derive Haplotype Blocks
and Measure Genomic Diversity in Populations Using SNP Genotypes 427

association studies. In genome-wide association studies (Hirschhorn et al. 2002, Pearson and
Manolio 2008, Kruglyak 2008), the premise is to test for associations between the variation in
a complex trait and causal mutations, however, for the most part we instead test for
association between the trait and a SNP in high LD with the causal mutation. Knowledge of
LD patterns has been shown to increase the power and decrease the amount of genotyping
required for association studies. For example, we can use information about LD and allele
frequencies across the genome to make informed decisions as to which SNPs (known as tag
SNPs) should be selected for the genotyping array. That is, the number of SNPs required in
GWAS can be reduced without a reduction in power if LD is extensive (Carlson et al., 2001).
Linkage disequilibrium is also used in the studies of a species genetic history and origins,
the detection of natural selection, and the biology of recombination from inferring the
distribution of crossover events from patterns of LD Pritchard (2001). In particular for
animal production, working out LD is important within breeds to determine the SNP
BOX 1

SNP: A single-nucleotide polymorphism is a DNA
sequence variation occurring when a single nucleotide
A, T, C, or G in the genome (or other shared
sequence) differs between members of a biological
species or paired chromosomes in an individual. For
example, two sequenced DNA fragments from
different individuals, AAGCCTA to AAGCTTA,
contain a difference in a single nucleotide. In this case
we say that there are two alleles: C and T. Almost all
common SNPs have only two alleles. (Source:
http://en.wikipedia.org/wiki/Main_Pag)

QTL mapping: Quantitative trait locus (QTL) mapping
means identifying genes that affects a complex
phenotype like disease or explains significant
proportion of genetic variation of a quantitative trait
observed in mapping population. It uncovers the
genetic basis of quantitative variation in a trait.

GWAS: A genome-wide association study (GWAS) is
an approach that involves rapidly scanning genetic
(SNP) markers across genome in hundreds of
individuals to find and quantify genetic variations in a
particular disease or trait associated with each SNP
screened. It uses highly dense SNP marker genotype
data (nearing 1 million in some animal species) to
detect association with phenotypes. These study
require larger sample sizes than QTL mapping and
requires validations in other independent populations.
GWAS techniques result in a panel of predictive
markers that can predict a future phenotype of an
individual. How good will be a prediction by a set of
markers depends on whether or not they are linked to
and/or in linkage disequilibrium with causal loci.

density for GWAS, and across breeds to check whether LD based predictions are expected to
persist between breeds.
To quantify the amount of LD, a variety of different statistical measures have been
proposed: D, D

and r
2
. D is the basic measure for LD and the formula is D = P
AB
P
A
* P
B

(where P
A
and P
B
are the marginal allele frequencies at two loci on a chromosome; and P
AB

is the probability of the observed haplotype). D equates to 0 if and only if the two loci are
independent. A disadvantage of D is that the range of possible values depends on the
marginal allele frequencies and therefore, as there is no standardisation, it is difficult to
compare D values. D is the standardisation of D and its formula is shown in Equation

max
0
D
D when D
D
= Where D
max
= the smaller of P
Ab
and P
aB

min
0
D
D when D
D
= < Where D
min
= larger of P
AB
and P
ab
Equation 1. Measuring LD using D for 2 loci A and B with 2 alleles.
The most widely used measure for LD is a correlation between pairs of biallelic SNPs
denoted by r
2
(refer Equation 2). Some of the properties of r
2
: a value of 0 implies
independence between the SNP alleles (perfect equilibrium); a value of 1 implies perfect LD.
Most pairs of SNP alleles have an r
2
greater than 0 or less than 1 indicating the strength of
the association between their alleles. An r
2
of 0.7 or 0.8 is considered strong LD between
SNPs. For the most part, the strength of the correlation between SNPs decreases as the
genetic distance between the SNP increases. The r
2
measure also has another useful
property; it is claimed to be related to the power of association mapping and can
consequently be used to estimate how large the sample size needs to be to capture
association (n
2
=

n
1
/ r
2
where n
1
is the number of cases and n
2
is the number of controls).
Currently for human genotyping arrays, tag SNPs are selected based on an r
2
concept of LD
structure for their pairwise ability to predict the genotype of untyped SNPs. For species with
limited knowledge of LD, the SNPs are selected evenly distributed.
2
2
* * *
A B a b
D
r
P P P P
=
Equation 2. Given haplotypes for 2 loci A and B with 2 alleles. Where P = allele frequencies,
and D is a basic measure of LD e.g. *
AB A B
D P P P = .
Population genetic factors that affect LD among specific groups of SNPs are numerous,
complex, and not clearly understood. Some of the acknowledged factors are mutation,
historical recombination, natural selection, founder effects, migration, population growth,
random drift, gene conversion, and population admixture. Only recombination is discussed
further in this chapter. It has been argued that recombination is one of the main factors
affecting LD (Ardlie et al., 2002). The rate of LD decay depends on the rate of recombination
and for the most part, decay in LD is affected by how close the alleles are together. Little is
known about the actual molecular mechanism of recombination and why some regions of
the chromosome experience more recombination than others. What we do know is that there
is variation in recombination rates, and regions of recombination appear and disappear over
evolutionary time. By studying the patterns of LD we can at least infer the distribution of
recombination events.
In the literature LD is intertwined with the term haplotype. There are many definitions of
the term haplotype in the literature, herein haplotype is used as being half of a genotype,
that is, a set of ordered SNP alleles on a single chromosome that are transmitted as one unit
from a parent to an offspring (Ardlie et al., 2002). Theoretically a haplotype, one unit, could
comprise any number of SNPs from only 2 SNPs to every single SNP on the chromosome. In
reality, however, recombination events result in haplotype blocks comprised of varying
numbers of SNPs.
Early studies of pairwise LD (i.e. using 2-locus haplotypes) observed complex patterns of
LD implying a random nature. It is now becoming clear that despite many generations of
segregation from a common ancestral chromosome, certain combinations of neighbouring
SNP alleles (haplotype units) have remained unchanged. In other words, there are stretches
of DNA that are almost never divided during meiosis (Gibbs et al., 2003). Although we do
not fully understand the biological processes that give rise to recombination in some regions
of the chromosome and not in others, there still appears to be some non-random
underpinning mechanism. More recently the International Hapmap Project (Gibbs et al.,
2003) has shown that the underlying structure of LD in a genome could be divided into
discrete haplotype blocks. Using evidence from their LD measures, a haplotype block
represents a region with a few haplotypes (2-4 per block) in a population separated by a
region with many haplotypes in the population. Their proposed haplotype block model of
LD, from a recombination perspective, is a region of high LD separated by recombination
hotspots. There are two popular methods for block definition: 1) using pair-wise
disequilibrium to define regions of high LD separated by recombination hotspots, and 2)
defining regions with high or low haplotype diversity.
1.2 Phasing SNP genotypes for deriving paternal and maternal haplotypes
We currently have the technology to observe genotypes but not haplotypes. That is, we do
not observe individual alleles on the chromosome. This immediately presents a problem for
haplotype analysis since the phase is not known when SNPs are heterozygous. For example,
given the genotype of 2 SNPs with homozygous alleles at 2 different loci, 11 and 22
respectively; the haplotype on both the paternal and maternal chromosome is conclusively
12. However, given the genotype of 2 SNPs with heterozygous alleles, 12 and 12; we
do not know which allele is inherited from which parent. The possible haplotypes are
shown in Figure 1.
We cannot say for certain which alleles on a haplotype go together when using genotype
data with heterozygous SNP alleles. Consequently we need to determine or infer the phase
from other methods. There are 3 possible methods available to the researcher: 1) use
pedigree information; 2) use molecular methods to single out individual chromosomes to do
genotyping (currently only possible on small regions; and 3) statistical methods to infer the
haplotype given genotype data. From literature, there are several algorithms and programs
for inferring haplotypes. Two of the most popular programs are called PHASE, which uses
algorithms based on Bayesian coalescent models Stephens et al. (2001) and fastPHASE,
which uses an EM algorithm and cluster model Scheet et al. (2006). The default PHASE and
fastPHASE output format has been adopted as the format required for the input data to
SNPpattern.


Fig. 1. Possible haplotype when 2 SNPs have heterozygous alleles.
PHASE is a statistical method inspired from coalescent theory. The coalescent theory in
essence is the tracing of alleles, shared in a sample of individuals from a population, back to
the most recent common ancestor Fu et al. (1999). This theory can predict the expected
patterns of haplotypes in natural populations. The PHASE method is Bayesian and uses the
a priori expectation of haplotypes to inform haplotype reconstruction (see Equation 3). The
phase reconstruction procedure is to evaluate the conditional distribution of the unknown
haplotypes corresponding to the genotypes for the individuals from a population sample.
PHASE uses Gibbs sampling (Kim, 2001) to obtain an approximate sample from the
posterior distribution of unknown haplotype pairs given genotype data (e.g. Pr (H | G) is
the posterior probability that the reconstruction of the haplotype pairs is correct, given the
genotypes and knowledge of previous haplotype reconstruction states). In the most
simplistic terms, the algorithm begins by estimating the haplotypes for a randomly chosen
individual on the assumption all other haplotypes are reconstructed correctly. The
algorithm reiterates the process enough times to result in an approximate haplotype
reconstruction from the posterior probability. Stephens[38] claims that PHASE, is
sufficiently accurate that reconstructing haplotypes experimentally, or by genotyping
additional family members, may be an inefficient use of resources.
) (
Pr( | ) Pr( )
Pr |
Pr( )
G H H
H G
G
=
Equation 3. Bayes theorem.
where,
Pr(H|G) is the conditional probability that the reconstruction of the haplotype pairs is
correct given the genotypes.
Pr(H) is the prior (unconditional) probability the reconstruction of the haplotype pairs is
correct irrespective of genotype data.
P(G) is the total probability of observed genotypes across all possible haplotypes (acts as a
normalising constant).

Where P = chromosome inherited from father; M = chromosome inherited from mother; Genotype
for SNPs = 12)
Pr(G|H) is the conditional probability of obtaining the genotypes given the haplotypes.
The fastPHASE software package is a statistical model that captures patterns of LD. The
variation in the patterns can be applied to estimate missing genotypes and to infer
haplotype phase in samples of unrelated individuals from natural populations from
unphased genotype data. The fastPHASE statistical model uses an approximate coalescent
with recombination prior manifested from the fact that over short genomic regions
haplotypes in a population have been observed to cluster into groups of similar haplotypes
because of recombination (Stephens et al., 2005). The model also considers each cluster of
observed haplotypes to represent a common haplotype and each haplotype is assumed to
have evolved from a single cluster. The membership of each cluster is allowed to change
along the chromosome in accordance with a hidden Markov model (Scheet et al., 2006). An
expectation-maximization (EM) algorithm (Dempster et al., 1997) is used to estimate the
model parameters.
The paper presents the development of SNPpattern as a simple bioinformatic tool to
rapidly screen the genome for haplotype structure, perform some basic descriptive
genome statistics and link interesting haplotypes to functional information. We have
tested our software SNP pattern on Ovine 60k SNPchip data (Goodswen et al., 2010). One
impetus for the development of SNPpattern was to understand the degree of diversity in
LD architecture between different livestock breeds (McKay et al. 2007). It was thought that
with our increased understanding we could potentially predict effect of genome selection
across breeds, which is based on SNPs being in LD with causal variants for the trait of
interest. In addition, we expect SNPpattern be used in the comparison of LD structure in
detecting and localizing genomic regions where selective sweeps
2
have occurred (Smith et
al., 1974).
2. Development of SNPpattern
A commonly used software package for computing LD statistics and haplotype patterns for
populations from genotype data is Haploview (Barrett et al., 2005). One of the interesting
features of Haploview, is its ability to generate haplotype blocks. Haploview has a number
of methods to partition the genome into blocks: 1) block definitions are based on D'
confidence bounds e.g. SNP pairs are defined to be either strong LD (.i.e. no evidence of
historical recombination) or strong recombination. The algorithm is taken from Gabriel et
al. (2002); 2) the block definition is based on a four gamete test of Hudson & Kaplan (1985)
proposed by Wang et al (2002). In brief, for each SNP pair, the population frequencies of the
4 possible two-SNP haplotypes are computed (e.g. SNP 1 = A/a and SNP 2 = B/b. The 4
haplotypes are AB, Ab, aB, and ab). If all 4 haplotypes are observed with a frequency >=
0.01 (a user definable threshold), a recombination is assumed to have occurred. If only 3
haplotypes are observed no recombination is assumed. A block is formed when there has
been no recombination for successive SNP pairs.
HaploBlock is a software package, which has as one of its capabilities the inference of
haplotype block models from phased or unphased data. It primarily uses a Markov chain
and can account for recombination hotspots, bottlenecks, genetic drift and mutations
(Greenspan & Geiger, 2004). HapBlock (a different program to the similarly named

2
A selective sweep can be caused when there is a strong directional selection for a favourable new allele
that increases its frequency. Alleles in close proximity to the new allele are swept to fixation.

HaploBlock program) provides both a parametric dynamic programming algorithm for
block partitioning with a fixed genome coverage using the minimum number of tag SNPs,
and a discrete dynamic programming algorithm for block partitioning with a fixed number
of tag SNPs that can cover maximum length of genome (Zhang, 2005). Finally, GERBIL is
another software package that implements an algorithm for simultaneously phasing
genotypes into haplotypes and block partitioning. It considers the phasing and the block
partitioning as a maximum likelihood problem and uses the EM algorithm to solve it
(Kimmel & Shamir, 2005). Table 1 shows a brief summary of the publicly available programs
that provide functionality to define haplotype blocks from genotype data.

Program Primary LD metric Visualisation of LD
PHASE/
fastPHASE
import
$$

Implemen-
tation
OS
Haploview D and r
2
Yes No Java Linux
Windows
HapBlock

D No No C++ Linux
HaploBlock

** No No Ansi C Linux
Gerbil ++ Yes No Java/C++ Linux
Windows
SNPpattern Pattern
frequency in block
No Yes Perl Linux
Windows
Table 1. Freely available programs providing haplotype block definition from genotype
data functionality.
LD = Linkage disequilibrium: OS = Operating System platform: ** A Bayesian Network
statistical model and Markov chain at its core: ++ Uses an expectation-maximization (EM)
algorithm: $$ imports genotype data in a PHASE/fastPHASE format without modification
In studies on human populations it has been shown that the human genome can be divided
into haplotype blocks (Gabriel et al., 2005). A haplotype block is an ancestrally conserved
region of varying size containing only a few common haplotypes in the population. The
haplotype blocks have discrete boundaries defined by recombination hotspots (Wall et al.,
2003) and Phillips et al., 2003) [51, 74]. SNPpattern implements a haplotype-block model as
an empirical approach to best describe the linkage disequilibrium (LD) patterns. From a
SNPpattern programming perspective, a haplotype block within a population is inferred
from a region on the chromosome where there is a low SNP allele pattern count for a
particular block size, separated by a region with a large SNP allele pattern count. It is
proposed that the block with a large count relative to other counts along the chromosome is
a region where more historical recombination events have occurred.
Whist the importance of pairwise measures of LD is acknowledged it may not always be the
most appropriate measure of how strong LD is across an entire region that contains many
SNPs. In particular, identifying precise haplotype-block boundaries may be difficult when
using r
2
. The r
2
measure produces for each pair of SNPs an LD strength estimate
fundamentally based on probability. There is no practical evidence to explain a difference in
the values of r
2
between other paired SNPs in adjacent and further away regions. Pairwise
measures of LD differ from SNP to SNP and defining haplotype blocks is especially open to
interpretation when r
2
values range between 0 and 1. There exists an uncertainty as to how
one LD estimate in one region relates to an LD estimate in another region because SNP pairs
are not necessarily independent (i.e. one region may functionally affect another region) and
consequently this diminishes the certainty of which SNPs belong to which haplotype block.
For example, there are cases where 2 SNPs exhibit strong pairwise LD but show different r
2

to a SNP in between, and a low strength pairwise LD is not necessarily indicative of high
ancestral recombination. In other words, SNPs in close proximity are not always in pairwise
LD and by contrast, SNPs far apart can be in pairwise LD (Phillips et al., 2003). We can also
expect the haplotype block boundaries to be different depending on the sample size and
SNP density used. Another limitation of r
2
, particularly for marker-assisted selection in
livestock, is that the r
2
can be the same between a SNP marker and a potential causal variant
in different populations, and yet the phase may be different (Roos et al., 2008). Deriving
clear information about the joint inheritance of alleles in a chromosome segment is also
expected not to be easy from r
2
measures. It is argued instead that we can infer the joint
inheritance of alleles from inferring which haplotype blocks were inherited, if we know
which haplotype blocks exist in a particular population i.e. we can make inference about
identity by descent (IBD) of alleles in particular regions. In light of some of these
shortcomings discussed, a multiple SNP allele block approach in preference to r
2
was
implemented through SNPpattern.
The required input data for SNPpattern is phased genotype data from either a single group
or multiple groups of individuals (e.g. from different animal breeds or subpopulations). The
premise for the multiple SNP allele block approach is to count the frequency of SNP allele
blocks, of different sizes, found in the genomes of the group members. For example, a block
of 5 SNPs spanning a few thousand base pairs could potentially comprise 32 different SNP
allele patterns if the SNPs were totally independent and the population was of infinite size
(the number of possible SNP allele patterns is 2
n
where n is the number of SNPs in the
block). The general process of the program is that it counts the frequency of the various
SNP allele patterns found in the same chromosomal location (the same SNP allele block
region) across each individual in the group sample; then repeats the process for the next
SNP allele block region along the genome, and so on. From the counts we can infer the
haplotype blocks after taking into account the population structure and allelic frequencies (a
user of SNPpattern also needs to be aware that there are numerous other population genetic
factors that affect LD and determine haplotypes). The inferred haplotype block represents a
region with a few distinct SNP allele patterns (indicating small amount of haplotype
variation) in a population separated by a region with many SNP allele patterns (indicating
an excessive amount of haplotype variation) in the population. In a typical short
chromosome segment, we can expect only a few distinct SNP allele patterns. Hence the
larger the SNP allele block size the less likely the distinct SNP allele patterns appear by
chance because of the increased probability of recombination over larger distances. It is
argued that the comparison of SNP allele pattern counts can be used as a measure of genetic
distance and this comparison forms the basis for a haplotype diversity analysis within and
between groups.
In addition to implementing the core components for the multiple SNP allele block
approach, SNPpattern also implements similarity scoring between individuals. We can
expect that the more the SNP allele patterns between two individuals are similar the more
likely they will have a similar haplotype structure. Taking this one step further, if two
individuals share the same extended SNP allele patterns over the same genomic region, the
chance that they carry the same causal variant allele relationship by descent is much higher.

Linkage disequilibrium mapping to identify the chromosomal region (the haplotype block)
containing a QTL has proven to be a powerful tool Barrett et al., (2005) and Hayes et al.,
(2006). However, once the haplotype block has been identified, LD provides no further
information to help localize the actual variants within the block (Rioux, 2001). It has been
proposed that advantageous mutations through directional selection are more likely to
occur in a region of low recombination (Wall et al., 2003). Conversely, there is evidence that
there are alleles in recombination hotspots that are more likely to initiate the double-strand
break associated with recombination (Jeffreys & Neumann, 2002). One of the outputs from
SNPpattern is a list of chromosomal start and end locations of SNP allele blocks identified to
have low and/or high haplotype diversity. In the program testing section of this chapter,
how this output list could be used to link these identified regions to genomic annotation is
demonstrated. We used the FunctSNP R package that we have developed earlier (Goodswen
et al., 2010) .To recover the biology role of genomic regions with low haplotype diversity, a
systems genetics or system biology approaches would be needed, as demonstrated in
Kadarmideen et al., (2006) and Kadarmideen (2008).
3. Implementation of SNPpattern
SNPpattern was written in the Perl programming language. The following sections describe
the methods and rationale that have shaped the development. We have tested SNP pattern
on Ovine 60k SNPchip data and these results are based on our earlier work (Goodswen et
al., 2010).
3.1 Input data
The default PHASE and fastPHASE output format has been adopted as the format required
for the initial input data to SNPpattern. Figure 2 shows the format and is described here as it
governs how the data are processed and is an aid to understanding the methods to be
described later.
The genotype data for each individual is represented by 3 rows. On the first row is a unique
identification of the individual. The second and third rows are the genotypes of the
individual. For each consecutive locus, one allele is entered on the second row, and one on
the third. SNPpattern expects that genotypes are phased such that the entire second row is
inherited from one parent and the third row from the other parent. It is also expected that
the alleles appear in the sequential order that they occur on the chromosome.

Fig. 2. Data input format for SNPpattern.
BEGIN GENOTYPES
# id 1
1 2 1 2 2 2 1 2 1 2 1 2 1 2 2 2 1 1 2 1
1 1 2 2 1 2 1 2 2 2 1 2 1 1 2 2 1 2 1 2
# id 2
2 2 1 1 2 1 2 2 2 1 2 1 2 1 2 1 2 1 2 2
1 2 1 2 2 2 1 2 1 2 1 2 1 2 2 2 1 1 2 1
# id 3
2 2 1 1 1 2 2 1 2 1 2 2 2 1 2 1 2 1 2 2
1 2 2 1 1 2 2 1 2 1 2 2 2 1 1 1 2 2 2 1
END GENOTYPES
3.2 Grouping data
In its simplest form, SNPpattern will accept an input, such as the one shown in Figure 2, and
treat all individuals as members of the same group. The output will consequently be results
for haplotype diversity within a group. The results will also be for the entire genome
without any reference to the chromosomal location of the haplotypes. In spite of this, it is
expected (although not mandatory) that an additional file be provided as input, which
contains phenotypic information about the individual. Table 2 shows as an example the first
9 lines of a fictitious phenotype file and in this instance one specific to livestock species.
Grouping data is obviously an essential part of the evaluation of haplotype diversity between
groups. It is also a hugely critical part to account for the count biases that may be introduced
due to population structure. For example, if in a particular sire breed group the number of
progeny from each sire is disproportionate then the SNP allele pattern count will be biased
in favour of the progeny with the largest number of siblings. Grouping an equal number of
progeny from each sire should prevent the bias. SNPpattern includes the functionality to
group the genotype data of individuals according to user-defined criteria specific to
information held in columns in a phenotypic file. Theoretically the program can create a
group based on any combination of columns when using the AND Boolean logic. For
example, group all individuals according to sire breed AND year of birth. Separate output
files for each group criteria are generated containing the genotype data of the group
members. The output format is the same as that shown in Figure 2. The program also allows
the user to use comparison operators (=, >=, <=, >, <) on any combination of column criteria.
For example, if we want to group all the female progeny in area 03 born after the year 1972
having a particular parent ID, the equivalent pseudo code is sex = F AND Area = 03 AND
Year of Birth > 1975 AND parent ID = 433. The grouping of the data is of course at the
discretion of the researcher to create genetically meaningful groups. Summary information
about the groups can also be generated. SNPpattern provides the flexibility of the grouping
through a configuration file in an INI file format.
Another optional file that can be provided as input is a SNP mapping file. Such a file allows
the contents of a group file to be divided further into separate chromosome files. This
division of the genotypes for the entire genome into their respective chromosome locations
allows for the comparison of haplotype diversity of a particular chromosome in one group
with the same chromosome number in another group. The fact that selective sweeps act
differently in different chromosomes is one example as to why a study of haplotype
diversity may be needed on a chromosome basis (Montpetit & Chagnon, 2006). It is
mandatory that the SNP mapping file contains the SNP location and the chromosome
number on which it resides. SNPpattern expects the SNPs in the file to be in the order that
they are located on the chromosome. It is also an expectation that the SNP mapping file is
most likely obtained from another source and will contain redundant information to
SNPpattern. Therefore another configuration file, specific to dividing the genome into
chromosomes, allows extraction of only the required SNP location and the chromosome
number without the need for the researcher to modify the SNP mapping file. It may be
arguable as to why a separate file is created for each group and/or each chromosome sub-
group. From a programming perspective separate files are created for 3 reasons: 1) the
output file format used is the same as PHASE and fastPHASE. It is envisaged that the
SNPpattern group files could be imported into other programs that use this same format; 2)
the separate files are a permanent record of the grouping that can be reused, as opposed to
temporary grouping only at runtime; and 3) the data files can be extremely large and slower
to parse the content if all groups are recorded separately but in the one file.

Human ID Parent ID Region Sex Parent ethnicity Year of birth Body Weight (kg)
1 330 01 F American 1978 74.6
2 330 01 M American 1971 99.0
3 405 02 F African 1970 77.4
4 405 02 M African 1975 63.6
5 433 03 M Asian 1972 79.0
6 433 03 M Asian 1971 67.0
7 433 04 F Asian 1979 73.0
8 405 05 F European 1974 97.4
9 405 05 M European 1976 94.0
Table 2. Example contents of a phenotypic file.
3.3 Multiple SNP allele block approach
This section describes the multiple SNP allele block approach implemented through
SNPpattern. We have tested SNP pattern on Ovine 60k SNPchip data and tables 3-6 are based
on our already published work (Goodswen et al., 2010). With reference to Figure 2 the 2
rows of biallelic SNPs contained within the phased genotype file are extracted (in this
instance, a single 1 or 2 constitutes a SNP allele). One row represents the SNP alleles
inherited from one parent, and the second row represents the SNP alleles inherited from the
other parent. So in effect, we have a representation of paternal and maternal chromosomes
composed of a long serial SNP allele pattern of 1s and 2s. Without prior knowledge, the user
will not know which row represents which parental chromosome. However, when the SNP
allele pattern analysis progresses the identity of the row representation may become
apparent as will be demonstrated in the program testing section.
The underlying unit of the multiple SNP allele block approach is of course the SNP allele
block. The serial SNP allele pattern from one row (e.g. representing the chromosome
inherited from the paternal side) is divided into block sizes of any specified number of SNP
alleles at the discretion of the researcher e.g. 3, 5, 10 or 100 (or larger) SNP alleles per block.
Then if required, the SNP allele pattern from the other row is divided into blocks of the
same specified size. Figure 3 shows the first 40 numbers of a SNP allele pattern of 1s and 2s
that represents either a paternal or maternal chromosome for one individual. In this
example, the entire SNP allele pattern is divided into blocks of 3 e.g. the first 3 blocks are
112, 212, and 211. For a n SNP allele block there are 2
n
possible SNP allele pattern
combinations of 1 and 2. Therefore, a 3 SNP allele block has 2
3
possible patterns (111, 112,
121, 122, 211, 212, 221, and 222).
For each SNP allele block along the row that represents either the paternal or maternal
chromosome, we count how many individuals in the group have the same SNP allele
pattern. For example, at block location 1 (Figure 3) we count, for each of the 8 possible SNP
allele combinations, how many individuals have the SNP allele pattern 111, then 112
etc. Table 3 shows an example of the SNP allele pattern count at the first 3-SNP allele block
along a paternal chromosome. We could expect an equal chance of observing any one of the
8 possible SNP allele combinations (assuming the SNP allele frequencies were equal) if there
was no underlying association between the 3 SNPs in the block. In reality however, we have
a SNP allele pattern count profile which is a result of many generations of random and non-
random SNP inheritance from a common ancestor. A challenge is to determine which of the
8 possible SNP allele combinations exist because the 3 SNPs were inherited by descent from
a common ancestor and which SNP allele combinations exist by chance alone. For long SNP

Fig. 3. Consecutive 3-SNP allele blocks along 1 row representing either a paternal or
maternal chromosome.
allele patterns (e.g. 10 or more SNP alleles per block)
3
that can be inferred to be a haplotype
block, identity is more likely by descent. For short SNP allele patterns (e.g. 3 SNP alleles per
block) inferred to be a haplotype block, it is more likely identical by chance. Nevertheless,
we can statistically test whether the observed count distribution has arisen from
independently segregating SNPs. On the other hand, it is debatable whether the test will
achieve the desired results. If SNPs are very close then we would expect SNPs not to segregate
independently, and the observed counts arise more from genetic drift (i.e. some SNP allele
patterns are more frequent due to limited population size and the large effect of the
contribution of only some ancestors to the current population). Despite the latter concern, in an
attempt to meet this challenge, expected and observed counts are still tested for statistical
significance. To determine the expected SNP allele pattern count, SNPpattern computes the
SNP allele frequencies (Table 3). For example, the expected proportion for SNP allele pattern
111 based on the allelic frequencies of each of the 3 SNPs and assuming independence is
0.072 ( Pr(SNP 1 allele 1) * Pr(SNP 2 allele 1) * Pr(SNP 3 allele 1)). The expected count for SNP
allele pattern 111 is therefore 72 (proportion expected * number of individuals).
Based on the allele frequencies, the null hypothesis is that we expect the observed and
expected count to be the same, and as a consequence the SNPs to be independent (i.e. SNPs are
segregating independently). A Fisher's Exact Test
4
for count data is applied as a statistical
significance test for each SNP allele pattern. Table 5 shows an example of how the data for
SNP allele pattern 111 from Table 4 is used in a 2 * 2 contingency table to compute the exact
probability of observing a table with this result (Equation 4). The p-values are obtained
directly using the hypergeometric distribution. The p-values (examples shown in Table 3) are
used as the conditional criteria to determine which SNP allele patterns were most likely to
have occurred by chance. In this example, the low p-value for pattern 111 indicates that the
hypothesis is unlikely to be true and therefore the SNPs within the pattern are not
independent. The success as to whether the challenge was met of distinguishing SNPs in a
haplotype block from SNPs in a random pattern is reviewed in the discussion section.
From a SNPpattern implementation perspective, some difficulty was encountered in
programming Fisher's Exact Test. A Perl module (Text::NSP::Measures::2D::Fisher2)
downloaded from CPAN
5
is currently being investigated for its suitability. As an interim

3
Dependent on the chromosomal distance between SNPs
4
Used in preference to Chi-Square test since expected counts may be less than 5
5
The Comprehensive Perl Archive Network

112212211111111211211211222121212
2111222 continued
1 2 3 4
5
SNP allele
blocks

SNP allele
pattern
Observed SNP
allele pattern
count
Expected SNP
allele pattern
count
Proportion
Observed
Proportion
expected
p-value
$$

111 14 72 0.014 0.072 6.07e-11
112 32 27 0.032 0.027 0.598
121 699 652 0.697 0.650 0.097
122 241 242 0.240 0.241 1.000
211 0 1 0 0.001 -
212 0 0 0 0.000 -
221 17 7 0.017 0.007 0.062
222 0 2 0 0.002 -
Total 1003
++
1003
++
1.000 1.000
++
Number of individuals in group
$$
p-values are obtained directly using the hypergeometric distribution following a Fisher's Exact Test
Table 3. Example of SNP allele pattern counts at the first 3-SNP allele block along a paternal
chromosome based on Goodswen et al., (2010)
measure, the statistical programming language R (http://www.r-project.org/) was used.
SNPpattern can output a file containing a list of the observed SNP allele pattern counts per
block in the first column and the expected SNP allele pattern counts per block (computed
from the individual SNP allele frequencies) in the second column. The output file can be
read directly into R and used as input to the function fisher.test () to conduct the Fisher's
Exact Test for count data.

SNP 1 SNP 2 SNP 3
Count Count Count
Allele Row 2
++
Row 3 Freq.
$$
Row 2 Row 3 Freq. Row 2 Row 3 Freq.
1 986 993 0.99 46 161 0.10 730 733 0.73
2 17 10 0.01 957 842 0.90 273 270 0.27
Total 1003 1003 1.00 1003 1003 1.00 1003 1003 1.00
++
Row 2 and Row 3 are the rows that represent the genotype data for each individual (refer Figure 4-1).
For genotype at SNP #1, 986 out of a total of 1003 individuals have a 1 on row 2, and 17 out of 1003
have a 2 a row 2
$$
Freq. = Allelic Frequency. For example, the population frequency of 1 at the SNP 1 location is (986 +
993) /2006 = 0.99. Likewise the population frequency of 2 at the SNP 1 location is (17 + 10)/ 2006 = 0.01
Table 4. Example of allele frequencies for 3 sequential SNPs.

Observed Expected Row totals
Pattern found 14
a
72
b
86
a+b
Pattern not found 989
c
931
d
1920
c+d
Column totals 1003
a+c
1003
b+d
2006
n
Table 5. A 2 * 2 contingency table for SNP allele pattern "111".
( ) !( )!( )!( )!
Pr( , , , )
! ! ! ! !
a b c d a c b d
a b c d
n a b c d
+ + + +
=
Equation 4. Fishers formula for exact probability of observing the data in a contingency
table.
Table 4 shows the SNP allele pattern count for the first 6 consecutive blocks for a 10-SNP
allele block size for a paternal chromosome. From the counts we can infer haplotype blocks.
As per the SNPpattern premise for a haplotype block previously described in the
introduction, it is a region with a low SNP allele pattern count separated by a region with a
large SNP allele pattern count. In other words, it is expected that if a block has a large SNP
allele pattern count relative to the counts within other blocks along the chromosome, it is
likely to be a recombination hotspot. For each paternal or maternal chromosome, SNPpattern
computes descriptive statistics such as the average number and standard deviation of
patterns found per block. A user definable count threshold can be applied to filter large
SNP allele patterns counts to infer the haplotype blocks. By default SNPpattern flags SNP
allele patterns with counts greater than 1 standard deviation above average. Of course, the
relevant count threshold to use and the interpretation of inferred haplotype blocks requires
thorough knowledge of group population structure. It is therefore critically important that
judicious grouping of genotypes takes place prior to the SNP allele pattern counts (refer
previous section Grouping data). Another point to note is that the chromosomal distance
between SNPs is not equal and therefore the physical size of the each block of SNPs is not
equal. Although SNPpattern computes and reports the physical block sizes, it does not adjust
the SNP allele pattern counts to compensate for unequal sizes.

SNP ALLELE BLOCKS
1 2 3 4 5 6
Pattern count 70 69 115 76 57 20
H/L flag
++
L L H L L L
Physical block size 619948 520686 437805 394152 398511 538789
++
H indicates block with SNP allele pattern count greater than user defined threshold; L indicates block
with SNP allele pattern count less than threshold
Table 6. SNP allele pattern counts per 10-SNP allele block along paternal chromosome.
In summary, for this section on the multiple SNP allele block approach, using SNP allele
pattern frequency counts as a measure, we can make comparisons between individuals,
groups of individuals, and groups. These comparisons then allow us to make informed
decisions about the general haplotype diversity. It is also expected that processing the same
genotype data several times using different block sizes, we can fine-tune the distribution of
the haplotype blocks. Finding similarity between individuals.
The method presented in this section was inspired from publications [80-83] on genetic
distance and similarity matrices. Two genetically identical individuals (i.e. identical DNA
sequences throughout the genome) will have identical haplotype structures. It therefore
could be argued that the more genetically similar two individuals are to each other, the
more likely they will have the same haplotype structure. In other words, the closer two
individuals are related the more the DNA sequences are expected to be in common. The
genotyped SNPs are of course not as accurate a unit of comparison as genome wide
nucleotide sequences. However, it is not unreasonable to assume that comparing the SNP
allele patterns between two individuals will provide a guideline as to the similarity of
haplotype structure. So, although this method does not show the actual haplotype structure,
the overall similarity in SNP allele patterns between individuals or groups of individuals
will give an indication of similarity in haplotype structure. As a simple example we take 3


Fig. 4. Method for scoring SNPs. It shows scoring of paternal chromosome (row 1) for only 2
out of 4 individuals.
ethnic groups (EG) called A, B and C. We then compare the SNP allele patterns for the full
length of the genome for each animal within each EG. From the comparisons we then
determine that EG A and B share the most similar SNP allele patterns. Therefore it is
proposed that there is a greater chance that EG A and EG B carry the same causal variant
allele relationship by descent than EGs A and C, or B and C. The comparisons are based on
similarity matrices whereby a score is incremented by 1 when a SNP allele of an individual
matches that in another individual (Figure 4). Each individual is processed in turn. In the
example only the paternal chromosome (row 1 in this case) is scored. The user can choose to
score either row 1 or row 2, or row 1 and row 2 of the phased genotypes contained in the
group input file(s).

ID A B C D Total
A 5 2 3 5 16
B 2 5 2 2 11
C 3 2 5 3 14
D 5 2 3 5 15
Table 7. Example similarity matrix. Shows a simple matrix constructed from made-up data
in Figure 4. Here we can see that individuals from ethnic groups A and D share the most
similarity; and individuals from ethnic groups A and B, and individuals from ethnic groups
B and C share the least similarity. An individuals overall similarity to all other individuals
in the group can be ranked according to its total similarity score. In this example,
individuals in A is considered the most similar and individuals in B the least. In practice the
similarity matrix is constructed from thousands of SNP allele pattern comparisons for
hundreds of individuals.
ID: Individual SNP allele pattern
A 1 1 2 1 2

ID: SNP allele patterns to compare:
Score
B 1 1 1 2 1 2
C 2 1 1 1 2 3
D 1 1 2 1 2 5

ID: Individual SNP allele pattern
B 1 1 1 2 1

ID: SNP allele patterns to compare:
Score
A 1 1 2 1 2 2
C 2 1 1 1 2 2
D 1 1 2 1 2 2

etc
3.4 Linking SNP allele block regions to genomic annotation
One of the output files from a SNPpattern Perl script is a file that contains all SNP allele
blocks where the number of distinct SNP allele patterns is low or high. The script allows
for a user-definable upper or lower pattern frequency threshold. For example, if a user
enters a threshold of <3 then only SNP allele blocks with a distinct SNP allele pattern
frequency of less than 3 will be output. Likewise, if the user enters >99 only SNP allele
blocks with a distinct SNP allele pattern frequency of greater than 99 will be output.
Figure 4-4 shows an example of the output file. The output consists of a list with 4
columns: Chromosome number of the chromosome containing the SNP allele block (the
genomic region of interest); start and end genomic location of SNP allele block; the
number of distinct SNP allele patterns found within the SNP allele block for a group of
individuals (only lists the genomic regions where the number of patterns is below or
above a user-defined threshold), and the average number of patterns per block. The
intended use of the output file is to act as a starting point for a researcher to find
biological meaning in regions identified to have low or high haplotype diversity.
Biological meaning may help in the understanding of why in some regions and not others
there is a conservation of the same alleles from generation to generation. In other words,
why is there only 1 or 2 distinct SNP allele patterns existing in the same genomic region
for all individuals in a group? Conversely, some regions have a large number of different
SNP allele patterns implying a hotspot region for recombination. Finding the underlying
biology within the hotspot region may provide clues to the mechanism of recombination.
The expectation is that the output list can be used for further downstream analysis such as
searching for annotation of the chromosome region within which the SNP allele block is
located.

Fig. 5. Example output file showing genomic regions with low SNP allele pattern counts.
As an example, we could find the genes within the genomic region. In the FunctSNP R
package (5) there is a function called getGenesByRange which returns the Gene ID for all
genes located between a user-specified start and end location.
3.5 Implementation summary
In summary, three sets of Perl scripts comprise SNPpattern: 1) grouping data scripts to
create separate data files for further downstream comparison analysis; 2) SNP allele block
scripts to find, count, and compare the SNP allele block patterns between any group of
individuals; and 3) similarity scripts - to score the similarity between individuals based on
an individuals entire SNP allele pattern. Table 8 encapsulates the primary function and
rationale of each script.
# Input file used: sire_31_match_10.txt
# Pattern Size: 10
# Haplotype row: paternal
# Chromosome No: Start of pattern: End of pattern: No. of Patterns:
Average No. per block
4 3000848 3170609 2 6.58
13 67400889 67687804 2 4.09

Perl Script
Name (.pl)
Primary function Rationale
Scripts for grouping and summarising genotype data
Group Genotype data is separated into files
according to a grouping criterion. For
example, the genotype of animals can
be grouped according to their sire
breed, or flock ID, or birth years.
Main purpose of dividing the data into
groups is to account for population
structure, facilitate the SNP-block
pattern counting within a group and
the comparison of the SNP-block
pattern count between groups.
divide Divide the bi-allelic SNPs in any group
input file (e.g. flock, breed, and sire
groups) into separate chromosome files.

Used as the main input for the SNP
allele pattern analysis scripts and in
particular for the multiple SNP allele
block approach
Scripts for finding, counting, and comparing SNP allele block patterns
derive_pattern Derive all SNP allele patterns of a
specified block size (e.g. 3, 100, 1000, 2000
etc.) that exist in the maternal and/or
paternal chromosomes for any group file
(e.g. either flock, breed, or sire)
Compiles all the unique SNP allele
patterns found in a group into 1 file.
Used as input to subsequent scripts to
find and count the frequency of these
unique patterns.
match Find and count the number of matching
SNP allele patterns found within a
specified block size along a paternal
and/or maternal chromosome for every
individual in a specified group.
An essential requirement for the
multiple SNP allele block approach
order_match Similar to match.pl except the output
is in a different format. Also creates a
group consensus file containing a
concatenation of the most common SNP
allele pattern found at each block. In
effect it creates a paternal or maternal
chromosome comprising the most
common SNP allele patterns in a group.
Enables a researcher to view and
compare, one block at a time, the SNP
allele patterns found within each block.
The group consensus chromosome can
be compared to the chromosomes of
individuals within the group and the
difference can be used as measure of
dissimilarity between individuals.
score Output the most frequent SNP allele
block pattern found at each block
location along the chromosome and
provide additional information such as
the percentage of animals with the
pattern, and chromosomal start and
end location of the block.
The most frequent SNP allele block
pattern is deemed to be the most likely
to be a haplotype. The statistics
provided may enable the researcher to
decide if the SNP allele pattern is a true
haplotype or one occurring by chance.

Scripts used to find similarity between animals based on SNP allele patterns
Sim For each animal in turn, list all other
animals in the same group in order of
SNP allele pattern similarity. The entire
chromosome is compared and
individuals are scored as to how many
SNP markers are the same.
Similarity matrices for individuals
within flocks, breeds, or sires can be
computed

Rank Similar to sim.pl except rank the
animals similarity to all other animals
in the group based on the summation
of the scores from the similarity
matrices.
Scores can be used as a measure of
genetic similarity between individuals
or groups. It is expected that similar
individuals will have similar LD
patterns.
Perl Script
Name (.pl)
Primary function Rationale
Miscellaneous scripts
SNP_map Count the number of SNPs per
chromosome and determine the
distance between each genotyped SNP.
Knowledge of the distribution and
distance between the genotyped SNPs
is important for interpreting haplotype
block boundaries.
pattern Generate a file listing all the possible
combinations of 1s and 2s given a
pattern size
Created as a general pattern generator
tool.
Table 8. The suite of Perl scripts collectively called SNPpattern.
4. Discussion
The SNPpattern program is a first version and is still in its development phase and the
program testing was a first attempt to analyse the haplotype structure within and across
populations. Nonetheless, SNPpattern in its current form will easily generate, with little user
required effort, output files that provide a researcher with information about LD and IBD
which can be used in population diversity and association studies. SNPpattern still has some
shortcomings that need to be addressed in future releases. Accounting for the population
structure of a group is currently at the discretion of the user by grouping genotypes
appropriately. During the grouping of genotypes SNPpattern allows specified animal IDs to
be excluded from the group e.g. if in a particular breed group the number of progeny from
each sire is disproportionate, animal IDs can be excluded to balance the proportions. It is
anticipated that knowing which animals to exclude may be difficult and the exclusions may
inadvertently introduce biases. Therefore a weighted SNP allele pattern count in accordance
to animal proportions may be a possible solution. Pritchard et al. propose a model-based
clustering method for using genotype data to infer population structure Pritchard et al.,
(2000). With this method it might be possible to assign individuals to appropriate groups
automatically. Another important omission that needs to be addressed is to take into
account, when interpreting haplotype block locations, the varying physical distance
between the SNPs within the blocks. Some SNPs are closer together in some regions and
further apart in others. Also, a sliding block window would improve accuracy and needs to
be implemented. For example, if we have a 3-SNP allele block the program currently uses a
window of SNPs from 1 to 3, 4 to 6, 7 to 9 etc. A sliding window would encompass SNPs
from 1 to 3, 2 to 4, 3 to 5 etc.
During the development of SNPpattern several statistical methods (in addition to Fishers
Exact Test for Count Data) were used in an attempt to determine which SNP allele pattern
has occurred because there is a correlation between the SNP alleles (possible members of a
haplotype block) and which SNP allele pattern occurred by chance. Despite taking allele
frequencies into account, no statistical test was found to reliably prove that SNPs were
inherited by descent. For example, let us suppose we have 3 SNP alleles in relative close
proximity to each other on a particular chromosome in a distant ancestor. Many generations
of progeny later, we have exactly the same 3 SNP alleles (the same haplotype block) in some
of the progeny. The challenge is to prove that these 3 SNP alleles where inherited from the
distant ancestor. The expectation is that these 3 SNP alleles have remained together on the
haplotype block because they reside in a genomic region which is involved in important

biological processes. That is, positive selection has ensured the survivable of the haplotype
block. Consequently it is expected that in a population of descendents from the distant
ancestor, the frequency of the haplotype block housing the 3 SNP alleles will be high within
the population. The increased frequency of the 3 SNP alleles might be explained by the
process of selective sweeps (Montpetit & Chagnon, 2006, Chevin & Hospital, 2008). A strong
selective sweep can result in only 1 or 2 haplotypes existing in the same region of the
genome for a population (Chevin & Hospital, 2008). Therefore, although further evidence is
required, it is argued that in some instances SNP allele patterns, which are overrepresented
in the population, indicate non-random SNP inheritance and could be considered a part of a
haplotype block. For example, there are cases where in a particular genomic region there is
only 1 out of 8 possible SNP allele patterns present in the population (i.e. 100% of
individuals have the same pattern). Many of the results from the Fishers Exact Test dispute
this argument. For example, in regions on the genome where nearly all individuals have the
same SNP allele pattern block and SNP allele frequencies on the block are high, Fishers p-
values indicate that the SNPs are independent.
Like all programs, the worth and accuracy of the output data from SNPpattern is totally
dependent on the data input. For example in the program testing on sheep breeds (Goodswen
et al. 2009), the frequency of SNP allele block patterns were counted and the similarity
between animals scored based on only 5,494 SNPs, which were genotyped for chromosome #1.
In other words, the interpretation of the LD patterns for chromosome #1 was based on the
state of 5,494 nucleotides. Chromosome #1 in fact is comprised of over 299,636,549 nucleotides
and, as in the case for sheep; there is an unknown number of SNPs. It is expected that as the
number of SNPs increase and the distance between the SNPs decrease the more the SNPpattern
outputs will be informative. Also it is important to know what selection criterion was used for
selecting the SNPs to be genotyped before interpreting the results obtained from SNPpattern.
For example, were the SNPs selected for even distribution across the genome and/or were the
SNPs selected as tags owing to prior knowledge of the LD structure. If the purpose of using
SNPpattern is to define haplotype blocks then it is expected that the results may be distorted if
the genotyped SNPs are tag SNPs.
This chapter solely focused on SNP haplotypes in the context of LD or selective sweeps due
to directional selection (natural or artificial) acting on the genetic variants affecting complex
traits measured / observed on the individuals. However, the consequences of this would
have been at the underlying biological level, namely the SNP haplotype diversity affecting
gene expression levels or protein abundance in cells and tissues of relevance to the complex
trait. This emphasises that future genetic studies on global gene expression patterns
(Kadarmideen et al., 2006 and Kadarmideen 2008) should be targeted at effects of LD /
expression-related SNP haplotype patterns. In fact, such studies could contribute to
prediction of transcription factor binding sites, using combined SNP and gene expression
datasets (Vonrohr et al., 2007). Further, identification of unique co-expression gene networks
and functional gene modules distinguishing different phenotypic extremes or case/controls
(e.g. Kadarmideen et al., 2011) could be speculated as being result of formation of distinct
SNP haplotypes after selective sweeps.
It is expected that in the very near future SNPs will, for the most part, be superseded by
entire DNA sequences due to the advent of low cost next generation sequencing (Hayden,
2009). With little modification, SNPpattern will handle DNA sequences in much the same
way as it currently does for SNP allele sequences (although the computer
performance/capability is an unknown entity). It is envisaged that varying block sizes of
DNA sequences will be compared and counted between individuals to determine the
structure and distribution of LD. Also, comparing entire DNA sequences between
individuals is perfect for determining genetic similarity.
Although the motivation for developing SNPpattern was to find patterns of LD, it is
suggested that common SNP allele patterns could be used in association studies (Botstein &
Risch, 2007). Common SNP allele patterns is only an interim suggestion, as it is expected
that using common DNA sequences in association studies will prove to generate the most
reliable results in the future.
5. Conclusions
We described the development of SNPpattern, which is the collective name for a suite of Perl
scripts essentially designed to group, count, and compare SNP allele patterns of various
block sizes. Differences in SNP allele block frequency are used as a measure of haplotype
diversity within and between groups. A SNP allele pattern represents SNPs inherited from
one parent and is a product from phased genotype data. The SNP allele pattern from a
programming point of view is simply a line of either 2 characters (0 or 1, 1 or 2, A or B)
representing 2 different states. The main factor that drove the development of SNPpattern
was the premise that studying SNP allele patterns can reveal useful information to help
understand the genetics of individuals within groups and across groups. The use of
SNPpattern has been illustrated on sheep breeds (Goodswen et al., 2009) but it is indeed
generic software meant for all species. SNPpattern allows researchers, given any phased
genotype data in a PHASE or fastPHASE format, to analyse SNP allele patterns within any
user-defined SNP allele block size. These SNP allele patterns can be compared between
user-defined groups. The primary objective of the tool is to provide a researcher with useful
information on SNP allele block patterns and as a major example of its usage, the
information can be used to quantify haplotype diversity within and between groups. While
there are similar bioinformatics tools that have a primary focus on haplotype inference
and/or analysis tools (such as Haploview, HapBlock, HaploBlock, and GERBIL) we have
found no tool that provides a smooth interface between a PHASE or fastPHASE output and
haplotype diversity/analysis.
Two main approaches for studying the SNP allele patterns have been implemented within
SNPpattern: a multiple SNP allele block and a pattern similarity scoring approach. For both
approaches, SNPpattern generates various descriptive statistics of the SNP allele patterns in
plaintext output files. It is not the authors intention to stipulate how a researcher should
interpret or use the information. Nevertheless, in this chapter suggestions were made as to
how SNPpattern might be used by a researcher. In particular, SNPpattern was proposed as a
generic tool for finding the patterns of LD using a multiple SNP allele block model. We have
demonstrated in another published paper how SNPpattern can be used to examine the
patterns and extent of LD within and between 4 Australian sheep breeds (Goodswen et al.,
2009). The results show that SNPpattern could be used to effectively evaluate overall
haplotype diversity within and between groups of individuals.
In closing, SNPpattern is a simple pre-screening tool to rapidly screen genome for haplotype
structure and provide insights on highly conserved biologically important haplotypes.
SNPpattern is implemented in Perl and supported on Linux and MS Windows. We have
tested SNP pattern on Ovine 60k SNPchip data (Goodswen et al., 2010). All scripts are
freely available from: http://web4ftp.it.csiro.au/ftp4goo17a/SNPpattern/SNPpattern.zip.

SNPpattern will be made available to the public via http://systemsgenetics.dk/pages/
resources.php in the future.
6. Acknowledgements
We would like to sincerely thank Julius van der Werf and Cedric Gondro for the inspiration
behind this paper and help with providing sheep SNP data for program testing.
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21
Algorithms for CpG Islands Search:
New Advantages and Old Problems
Yulia A. Medvedeva
Vavilov Institute of General Genetics, Russian Academy of Sciences,
Research Institute for Genetics and Selection of Industrial Microorganisms,
Russia
1. Introduction
CpG islands (CGIs) are regions having high GC and CpG content while generally
mammalian genomes are CpG-depleted. CGIs are often located in the promoter region of
the genes, mostly housekeeping but also tissue-specific. It is widely believed that CpG
dinucleotides within promoters CGIs are unmethylated and are targets for specific
regulatory protein binding. As a result, CGIs contain special sequence motifs for highly
affinitive protein binding (transcription factor binding sites, TFBS). Methylation of cytosine
in CpG context within such motifs could decrease the affinity of TF binding, increase the
attraction of methyl-binding proteins, affect the histones modification and, therefore, leads
to repression of genes transcription. The mechanism of local and global transcription
repression via CpG methylation is used in many different normal (development,
differentiation, aging, X-chromosome inactivation, imprinting) and pathological processes
(cancer and other diseases). However recently it has been reported that a class of normally
methylated but active promoters do exist.
Lately evidences of biological relevance of methylated CGIs or CGIs located far from gene
promoters appear. Such CGIs could act as regulator for pervasive transcription, which
seems to be actual genome feature rather than a side-effect of high-throughput techniques
errors. Replication origins are also reported to be associated with CGIs of any location.
As a consequence of specific nucleotide content, CGIs could affect DNA or RNA secondary
structures. For example, G
2-3
C
2-3
motif common within CGIs induces significant local curiosity
of DNA. Another motif, G-rich sequence (GRS) in 3 and 5 region of RNA, is known to form
specific structures, G-quadruplexes, on both end of RNA playing important role in its stability.
This motif corresponds to C-rich sequence in DNA, is likely to appear in CGIs.
Classical algorithms for CpG islands search use sliding window (SWM) or running sum
(RSM) and several distinct but not independent criteria (GC content, Obs/Exp
CpG
and
length). The thresholds for the criteria are rather arbitrary, unconcerned between species,
and demonstrate lack of biological interpretation. SWM algorithms are rather slow, RSM
algorithms are faster but tend to split large CGIs into several smaller ones and to omit CGIs
with nonuniform distribution of CpG dinucleotides along the sequence. Recently, several
different algorithms based on CpG dinucleotides clustering were implemented. Those
algorithms have smaller number of parameters and reasonable mathematical basics. The
comparison of the algorithms is tricky. Hypermutability of CpG dinucleotides lead to loss of


450
CGI conservation between species so comparative genomics cannot be applied for
estimation of the algorithms effectiveness.
To validate the results of CGI prediction authors use different biological and mathematical
properties. One of the most popular quality measures is the fraction of CGIs located near
promoters of protein coding genes and avoided overlap with Alu-repeats. This measure
couldnt be appropriate at least for two reasons. First, promoters of protein-coding genes are
likely to be a small fraction of all promoters as it became clear recently. Second, two classes
of promoters (CGI-dependent and CGI-independent) exist and their ratio is unclear.
Avoiding of repetitive sequences is more or less reachable for many algorithms, but now
authors prefer to remove Alu- repeats and other repetitive DNA sequences in advance.
Prediction of the methylation profile in different tissues in norm and in cancer is another
idea for validation. Algorithms of CGI search per se fail to predict correctly the distribution
of methylated cytosine in the genome. To distinguish between methylated and non-
methylated CGI machine-leaning techniques (MLT) are used. Those studies include
additional sequence features (di- and trinucleotide distribution, CpG and TpG frequencies,
TFBS, repetitive elements and others). Machine-leaning techniques are also applicable for
collecting promoter CGIs. The point that GC content and CpG frequency or density of CpG
clusters is not enough to describe special types of CGIs, is highly relevant. The main
problem of MLT approaches is that resulting model usually has a lot of parameters,
sometimes without clear biological meaning. Consistency of the models, build up by
different authors in the similar conditions is rather low, so those feastures could hardly be
used for CGI validation quality in general case.
A verification problem caused by lack of universal biological properties of CGIs results in an
absence of widely accepted definition. It should be mentioned that all algorithms trying to
predict CGIs with one particular function (promoter or unethylated CGIs) demonstrate a
high false-positive rate, probably due to the complex network of CGIs functions. Its
becoming clear that many different functional elements exist within one CGI. Moreover,
both methylated and unmethylated, both promoter and non-promoter CGIs seem to be
functional. So, one can conclude that contemporary algorithms for CGIs search based only
on GC and CpG content or on CpG clustering determine a chimeric class of objects.
2. Algorithms for CpG islands search
Nowadays, most popular algorithms for CpG islands search are still based on criteria
established more than twenty years ago (Gardiner-Garden & Frommer, 1987). The DNA
segment is considered to be a CpG island if it is not shorter than 200 bp, has GC content no
less than 0.5 and the ratio Obs/Exp
CpG
(1) no less than 0.6.
Obs/Exp
CpG
= N
CpG
*N/(N
C
*N
G
), (1)
where N
C
, N
G
and N
CpG
are numbers of C, G and CpG in the region of length N respectively.
Implementations of the basic idea vary in details, mostly in methods for search of the
segments having properties mentioned above.
2.1 Sliding window methods
There are several algorithms for CGIs search using sliding window methods (SWM):
CpGplot (Rice et al., 2000), CpG Island Searcher (Takai & Jones, 2002), CpG Island Explorer
(Wang & Leung, 2004) and CpGProD (Ponger & Mouchiroud, 2002).

Algorithms for CpG Islands Search: New Advantages and Old Problems

451
CpGplot represent the simplest variant of SWM. GC content and Obs/Exp
CpG
ratio are
calculated over a window of length 100 bp moving along the sequence with 10 bp steps.
CpG Island Searcher (usually referred to as Takai-Jones algorithm) uses a window of 200
bp moving along the sequence with 200 bp steps. It has an additional threshold for minimal
CpG dinucleotides in predicted CGI, equal to mathematical expectation of CpG
dinucleotides in Bernoulli sequence of given length and nucleotides probabilities, multiplied
by Obs/Exp
CpG
threshold. This feature lets authors exclude mathematical CGIs like 300 bp
sequence with 150 cytosines and one guanine in CpG context which fits standard CGI
criteria. This algorithm also merges two or more CGIs if they are spaced by less than 100 bp.
Takai and Jones also suggest using more strict thresholds of 500 bp for CGI length, 0.55 for
GC content and 0.65 for Obs/Exp
CpG
to find out CGIs associated with promoters of known
protein-coding genes and to avoid CGIs associated with Alu-repeats.
CpG Island Explorer is a modification of CpG Island Searcher from Takai and Jones. A
sliding window of CpG Island Explorer moves more slowly with a step of 10 bp. After
merging of close CGIs the resulting CGI is tested ones again to fit the criteria and if it does
not, one bp from each side is cutting until final CGI fits the criteria. Takai and Jones believe
that CGIs predicted by CpGIE are larger in length. Closely located CGIs are merged more
reasonably by CpGIE than by CpGIS.
CpGProD is a program dedicated to the prediction of promoters associated with CpG
islands in mammalian genomic sequence. In every sequence found by sliding window and
fitted the criteria of CGI the probability to find promoter is estimated as
p = exp(Z) / (1 + exp(Z)), (2)
where Z is linear combination of CGI length, GC content and Obs/Exp
CpG
. Also the
probability of a strand to be a template for transcription is estimated as in (2), where Z is
linear combination of AT- and GC-skews which are known properties of the nucleotide
sequence around the TSS. Coefficients for Z are estimated from two generalized linear
regressions trained with two datasets composed of CGIs obtaining and not obtaining TSS for
protein-coding genes or two datasets with different transcription templates in human.
2.2 Running sum methods
Running sum methods (RSM) were developed as an alternative to SWM. RSM try to find
segments of DNA having CpG dinucleotides more frequently comparing to the neighboring
genomic sequence. RSM work faster comparing to SWM. Initially RSM did not use CGI
criteria established in (Gardiner-Garden & Frommer, 1987). Most known methods from this
group are CpGreport (newCpGseek) (Rice et al., 2000) and unpublished algorithm of
Mikhlem and Hillier which is used in UCSC Genome Browser (http://genome.ucsc.edu)
and therefore became de facto a standard for CGI search.
CpGreport (or newCpGseek) scores each position in the sequence using a running sum
calculated from all positions in the sequence, starting with the first and ending in the last. If
there is not a CpG dinucleotide at a position, the score is decremented, if there is one, the
score is incremented by a constant value. If the score is higher than a threshold then a
putative CGI is declared. Sequence regions scoring above the threshold are searched for
recursively. It should be noticed that final CGI from predicted by this algorithm starts and
ends with CpG dinucleotide and doesnt necessary reach the initial CGI criteria (Gardiner-
Garden & Frommer, 1987). Authors found a lot of rather short CGI with high GC content
and CpG frequency and considered such CGI as overprediction (Rice et al., 2000).


452
UCSC CGI (Algorithm of Mikhlem and Hillier) is based on the RSM but include additional
check for CGI to fit the traditional criteria (Gardiner-Garden & Frommer, 1987). Total number
of CGIs obtained by UCSC is less than those obtained by CpGplot, as not every frame is tested
for fitting the criteria, but only those having score higher than a threshold on the first step.
CGIs predicted by the algorithm of Mikhlem and Hillier are often shorter from both ends
comparing to those predicted by CpGplot and also starts and ends with CpG dinucleotides.
2.3 CpG clustering methods
Next logical step of CpG searchers development is to implement actual CGI clustering
methods (CGCM). There are several such algorithms available: CpGcluster (Hackenberg et
al., 2006), CpG clusters (Glass et al., 2007), and CGI HW, an algorithm, developed by H. Wu
(Irizarry et al., 2009; H. Wu et al., 2010). These algorithms are based on segmentation of the
genome into regions with different frequency of CpG dinucleotides (CGI HW also uses
segmentation based on GC content). Unlike methods described above this approach to CGI
prediction is data-driven and allows finding CGIs in spices with different average GC-content
and CpG frequency.
CpGcluster has two separate steps: a CpG cluster search and an estimation of the probability
to find such a cluster by chance. Distance between neighboring CpG dinucleotides in random
sequence is simulated by geometric law with CpG frequency as a parameter. Hackenberg and
colleagues (Hackenberg et al., 2006) assume that within functional CpG cluster the distance
between neighboring CpGs is smaller than expected in random sequence. Authors show that
distances smaller than a median of the theoretical distribution is overrepresented in human
genome. The median distance between neighboring CpG (23-53 bp depending of the
chromosome) is used as a threshold, so each cluster consists of CpGs located no farther than
the threshold. All resulting CGIs start and end with a CpG dinucleotide. Each cluster has a p-
value calculated based on negative binomial distribution. Only clusters with p-value less than
1.0e-5 (1.0e-20 in (Hackenberg et al., 2010a)) are considered as CpG islands. Authors find about
200000 CpG islands in human genome (25000 CpG islands using the p-value threshold equal
to 1.0e-20). A lot of such CpG islands are shorter than 200 bp. Yet, authors show functionality
of some short CGIs and call them CpG islets (Hackenberg et al., 2010a).
CG clusters annotation also has two steps. The location of every CpG dinucleotide is
extracted from genomic DNA sequences. Using these positions, every overlapping sequence
fragment containing a fixed number of CpGs and having variable length is identified. For
each number of CpGs, the frequency of each fragment length is recorded. The threshold for
each maximum fragment length is defined as a local minimum in the fragment length
histogram, estimated by identifying zero values of the first derivative of a cubic spline fit.
Mapping the CpG-dense fragments back to the genomic sequence produces an annotation
track there each annotated locus is a conglomeration of one or more overlapping fragments
of variable length. As the basis for choosing the optimal track the number of overlapping
fragments at a locus normalized by the maximum fragment length is used. A track with
maximal fragments overlap per locus is selected based on genomic averages of this metric
for different numbers of CpGs per fragment. This approach allows authors to choose the
species-specific optimal number of CpGs per fragment for the final annotation.
CGI_HW (Algorithm of H. Wu) assumes that each chromosome is divided into 3 states: Alu
repetitive elements, baseline, and CGI. Alu-repetitive elements are removed in advance.
Hence, authors characterize the problem as that of a semi-HMM, with a known state for Alu
repetitive elements, so they consider the 2-state chain conditional on being in a non-Alu


453
state. Authors use the number of C, G, and CpG in segment of length L as parameters for the
model. Hidden state Y(s) for segment is 1 for CGI and 0 for baseline. Authors assume that
Y(s) is a stationary first-order Markov chain. The choice of the state is based on two HMM.
One is for GC content to be high or low with assumption of the binomial distribution
approximated with the normal density for baseline. The second one is for CpG number with
assumption of Poisson distribution for baseline. The length L=16 for the segment was
chosen based on the association of CGI with epigenetic marks. The approach summarizes
the evidence for CGI status as probability scores. This provides flexibility in the definition of
a CGI and facilitates CGI search in different species.
3. Validation problem
Having several methods for CGI prediction one is still unable to select the best one. The main
reason is the lack of validation criteria. Su and colleagues (Su et al., 2009) propose cumulative
mutual information of CpG dinucleotides as a measure of CGIs quality and show that its a
powerful criterion to avoid CGIs associated with Alu-repeats. Despite the power of this
mathematical criterion, most of the authors try using biological features for CGIs validation.
3.1 Sources for biologically relevant validation: DNA methylation and protein binding
Very first work mentioned CG-rich islands (Bird, 1986) considers them as DNA regions where
cytosine is unmethylated. Cytosine methylation usually appear in CpG context and increase
the probability of its deamination about 10-times (Ehrlich & Wang, 1981), leading to
enrichment of TpG and depletion of CpG dinucleotides in DNA. Absence (or decreased level)
of cytosine methylation within CGI is usually considered as an origin of CGIs in mammalian
genomes (Cross et al., 1994; Eckhardt et al., 2006). Modern research shows that methylated
cytosines within CpNpG are also targets for spontaneous deamination (Cooper et al., 2010).
No doubts, that cytosine methylation plays important role in CGI functioning. During early
development waves of methylation-demethylation generate tissue-specific genomic
methylation profiles. These profiles are stable in somatic cells generations due to replication
dependent maintenance methylation system (Brero et al., 2006). About 70-80% of cytosines
in CpG context are methylated in differentiated cells (Baylin et al., 1998), recent study shows
that cytosine is also methylated within CpHpN context (where H = , or ) especially in
embryonic stem cells (Baylin et al., 1998). Cytosine methylation influence DNA structure by
facilitating Z-from conformation (Behe & Felsenfeld, 1981), it also affect protein binding to
DNA, so most transcription factors (TF) usually bind unmethylated DNA.
There is a class of proteins (e.g. MeCP1/2, MBD1-6, SRA, and Kaiso) binding exclusively
methylated DNA (Saito & Ishikawa, 2002). MeCP1 protein complex binds methylated
cytosine using MBD2 protein (Berger & Bird, 2005) and also includes chromatin remodeling
complex NuRD/Mi2. MeCP2 is the key and well-studied member of methyl-binding
domain (MBD) protein group (Fatemi & Wade, 2006). Besides methyl-binding domain it
contains transcription repression domain (TRD) (Dhasarathy & Wade, 2008) and is involved
into DNA methylation establishment with DNMT1 (Kimura & Shiota, 2003). There are
evidences that both MeCP2 and MBD1/2 binds not just
5m
CpG but more complicated DNA
motifs, MeCP2 binds
5m
CpG with adjacent (A/T)
4+
, which is not true for MBD1/2 proteins
(Klose et al., 2005). MeCP2 binds DNA with higher affinity than MeCP1 complex leading to
more stable repression of transcription. For MeCP2 binding single
5m
CpG dinucleotide is
enough whereas MeCP1 complex needs dense clusters of
5m
CpGs (Ng et al., 1999).


454
Another well-known group of methyl-binding proteins consists of Kaiso and ZBTB4/33.
They obtain zinc-finger domain and bind DNA in sequence-specific manner. Data on Kaiso
binding site are controversial. Van Roy and McCrea (van Roy & McCrea, 2005) believe that
Kaiso binds
5m
CG
5m
CG. Sasai and colleagues (Sasai et al., 2010) assume that
5m
CG
5m
CG
motif is a place where two Kaiso molecules bind, one on every strand. The motif also has to
be in specific sequence environment. Its also known that Kaiso binds TNGCAGGA motif
having non-methylated cytosine, but with 1000-times lower affinity (Daniel et al., 2002).
There are some evidences that Kaiso is a global repressor of methylated genes and is
essential for early embryonic development. ZBTB4 protein binds CYGCCATC motif as well
as M
5m
CGCYAT (Sasai et al., 2010). It also has been shown that proteins of this group
demonstrate affinity to half-methylated DNA (Sasai et al., 2010).
Some other proteins also bind methylated DNA. CpG methylation of the CRE-motif
(TGACGTCA) enhances the DNA binding of the C/EBP (Rishi et al., 2010). UHRF1 and
UHRF2 (SET- and Ring finger-associated proteins, SRA) bind hemimethylated CpG and the
tail of histone H3 in a highly methylation sensitive manner and help assemble histones and
DNA into a nucleosome after replication (Hashimoto et al., 2009).
3.2 Sources for biologically relevant validation: DNA methylation and gene
expression
Nowadays there are two main hypotheses explaining DNA methylation origin during
evolution. Some authors believe that methylation system arose to inactivate viruses and
transposons (Walsh et al., 1998). Despite some evidences in favor of this hypothesis, most of
the authors nowadays suppose that main function of DNA methylation is a control of gene
expression during development and cell differentiation, most likely by influence on affinity
of different protein binding.
Promoter regions of many genes are unmethylated and demonstrate resistance to increasing
concentration of methylating agents (Bestor et al., 1992). Yet if promoter region become
methylated this usually leads to stable in cell generations and irreversible gene suppression
(Razin & Riggs, 1980; Schubeler et al., 2001). However some genes demonstrate rather high
expression independently to methylation level of their promoters (Shen et al., 2007) and
some promoters need to have methylated cytosine to be activated (Rishi et al., 2010).
Cytosine methylation affects transcription both directly by changing the affinity of TF
binding to DNA and indirectly by forming inactive chromatin domains. Both
5m
C and T
change DNA conformation in core positions of TFBS. For transcription repression in some
cases its enough to have one cytosine methylated, in other cases the level of expression is
correlated negatively with methylation level, but is independent on the exact position of
cytosine to be methylated. Inhibition of transcription caused by partial DNA methylation
can be overpassed by enhancers (Hug et al., 1996), however fully methylated promoters
cant be reactivated that way (Schubeler et al., 2001).
The possibility of active demethylation is still under discussion (S. C. Wu & Zhang, 2010).
Cytidine deaminase AID could play a role in this process in mammals (Fritz & Papavasiliou,
2010). Recently it has been shown that elongation complex also can participate in
demethylation (Okada et al., 2010). Even DNA methyl-transferases DNMT3a/b could force
cytosine deamination leading to reparation of T-G mismatch pair into correct C-G pair with
GC-biased reparation system (S. C. Wu & Zhang, 2010). Overexpression of MBD3 could also
play a role in demethylation (S. E. Brown et al., 2008). Yet active demethylation after
implantation of the embryo is very rare occasion (S. C. Wu & Zhang, 2010).


455
Different tissues and cell types demonstrate specific cytosine methylation patterns (Ushijima
et al., 2003), those patterns in the same tissue of different individuals are similar (Lister et al.,
2009), but not identical (Bock et al., 2008). Now a lot of regions with tissues-specific
methylation profiles (tDMRs) are known (Rakyan et al., 2008; Brunner et al., 2009;
Straussman et al., 2009; Xin et al., 2010). DMRs are likely to be involved in gene imprinting
(Lopes et al., 2003). Differential activity of imprinted alleles of the gene is dependent on
methylation of promoters, enhanserses or silencers of those genes (Li et al., 1993).
Females have one of the chromosomes inactivated in somatic cells (Gartler & Riggs, 1983).
The process of inactivation starts at early embryo stage with Xist activation (S. D. Brown,
1991), which leads to chromatin modification and methylation of promoters of most
(Deobagkar & Chandra, 2003) but not all (Zeschnigk et al., 2009) genes. Methylation and
gene repression profile of inactivated X chromosome is stable in cell generations.
Defect of normal methylation profile is a distinctive feature for different pathology
conditions (Ratt syndrome, psychopathologies (Egger et al., 2004), autoimmune diseases
(Richardson, 2007), hypertension (Frey, 2005)). Despite many evidences on epigenetic
changes in pathologies, cancer is the most known disease having abnormalities in
epigenetics, especially in DNA methylation (Jones & Baylin, 2002; Laird, 2003; Herrera et al.,
2008). Tumor cells demonstrate a lot of modifications in epigenetics status: general
demethylation of the genome, influencing chromatin structure, increased DNA methyl-
transferase activity, and hypermethylation of promoter regions of many genes resulting in
their repression. High probability of
5m
C to mutate into T brings about a lot of cancer-
specific mutations. Its importation to notice, that pathological profiles of methylation often
depend on environmental conditions and are inherited (Liu et al., 2008).
3.3 Sources for biologically relevant validation: CpG islands as promoter regions
The RNA polymerase II core promoter contains DNA motifs directing transcriptional
machinery to the transcription start site (TSS). Nowadays four DNA motifs are known to be
a part of core promoter: the TATA box, the TFIIB recognition element (BRE), the initiator
(Inr), and the downstream promoter element (DPE) (Kutach & Kadonaga, 2000). The TATA
box is an A/T-rich sequence, located about 20-30 nucleotides upstream of the TSS, that
binds TFIID complex (Burley & Roeder, 1996). The BRE having the consensus SSRCGCC, is
located immediately upstream of the TATA element in some promoters and increases the
affinity of TFIIB binding (Lagrange et al., 1998). The Inr was originally a motif encompassing
the TSS that is sufficient to direct accurate initiation in the absence of a TATA element
(Smale, 1997). Inr elements are, however, present in both TATA-containing and TATA-less
promoters and play a role in TFIID binding (Chalkley & Verrijzer, 1999). In mammalian
promoters, the Inr consensus sequence is RRA
+1
NWRR, where A
+1
is the TSS (Bucher, 1990).
The DPE acts cooperatively with the Inr helping TFIID binding and accuracy of
transcription initiation in TATA-less promoters (Burley & Roeder, 1996). The DPE is located
about 30 nucleotides downstream of the TSS and contains a common GWCG sequence
motif.
Saxonov and colleagues (Saxonov et al., 2006) demonstrate that human genes have two
different promoter types: AT-rich and GC-rich (associated with CGIs). They are easily
distinguishable not only in AT- or GC content, but also in different motifs overrepresented
in each promoter type. One can see that most of core promoter elements are GC-rich and
could be a part of a CGI-associated promoter. CGIs are often located in 5' regions of genes,
mostly overlapping with TSS (Gardiner-Garden & Frommer, 1987; Davuluri et al., 2001;


456
Ponger et al., 2001), and participate in regulation of transcription initiation (Rozenberg et al.,
2008). Housekeeping genes tend to have CGI promoter more frequently comparing to
tissue-specific genes (Zhu et al., 2008). However promoters of tissue-specific genes related to
development and embryogenesis are usually located in proximity to CGIs (Robinson et al.,
2004).
Many authors believe that CGIs exist since CpG dinucleotides inside them are protected
from methylation. The mechanism of such protection is assumed to be protein binding at
CGIs boundaries as it has been shown for Sp1 in the promoter of mouse aprt gene (Macleod
et al., 1994). Later role of Sp1 in CGI boundaries formation has been shown for other genes
(Tomatsu et al., 2002). Sp1 is often associated with CGIs as one of the key features (Macleod
et al., 1994; Rozenberg et al., 2008). In one of the first works on CGI (Gardiner-Garden &
Frommer, 1987) it has been shown that CGIs obtain many G/C-boxes (GGGCGG), which act
as a core for Sp1 TFBS (Briggs et al., 1986). Sp1 binds both methylated and unmethylated
DNA (Holler et al., 1988). Fan and colleagues (Fan et al., 2007) assume that all proteins with
zinc-finger domain can play a role in CpG boundaries formation. Some other proteins, like
VEZF1 (Dickson et al., 2010) and CTCF (Filippova et al., 2005; Recillas-Targa et al., 2006),
also participate in this process. Naumann (Naumann et al., 2009) shows that loss of such a
boundary (in fragile X-chromosome syndrome) leads to spread of methylation and gene
inactivation. Moreover CGIs obtaining CTCF binding sites can themselves play a role of
insulators forming boundaries of chromatin domains (Filippova et al., 2005).
Other DNA binding proteins with GC-rich binding sites can also decrease the level of DNA
methylation (Lin et al., 2000; Recillas-Targa et al., 2006). Its most likely that unmethylated CpG
islands form open chromatin structures simplifying the transcription (Choi, 2010). Binding
sites for Cfp1 (Thomson et al., 2011), E2F (Weinmann et al., 2002), ETS, NRF-1, BoxA, CRE, E-
Box (Rozenberg et al., 2008), p53 (Zemojtel et al., 2009) was found within CGIs.
Besides TFBS other DNA motifs are associated with CGI promoters. GC-skew, a feature of
all unidirectional promoters, is stronger for genes starting within CGIs than for genes
lacking this property (Polak et al., 2010). Tandem or simple repeats are also found within
CGIs (Hutter et al., 2006). Sequence motifs G
2-3
C
2-3
, typical for CGI, induce local DNA
curiosity and form G-qudruplexes at 5 and 3 ends of RNA molecule. G-quadruplexes in
DNA restrict methylation of CpG dinucleotides genome-wide (Halder et al., 2010).
3.4 Sources for biologically relevant validation: CpG islands located far from
promoter regions
At least 25% of CpG islands are located far from gene promoters (Ponger et al., 2001).
Although a lot of such CGIs overlap with repeats, (Graff et al., 1997; Ponger et al., 2001),
other CGIs dont (Ponger et al., 2001; Hackenberg et al., 2006). They are often located near 3'
gene region (Gardiner-Garden & Frommer, 1987) or within the gene (Hackenberg et al.,
2006). Such 3and intragenic CGIs are subject for natural selection not only on the protein
level, but also on the level of nucleic acids, which confirms their functional significance
(Medvedeva et al., 2010).
Many of CGIs located far from promoters of protein-coding genes perform important
biological functions. For instance, a CGI within intron 10 of KCNQ1 acting as a promoter of
antisense RNA transcript is involved into imprinting regulation of the locus (Smilinich et al.,
1999). Imprinting of MAP3K12 gene is caused by differential methylation of a CGI located in
its last exon (Takada et al., 2000). Many CGI around the 3' ends of genes affect its expression
in normal tissues (Appanah, Dickerson et al. 2007) and in cancer (Shiraishi et al., 2002).


457
Intergenic methylation plays an important role in regulation of alternative promoters
(Maunakea, Nagarajan et al. 2010), modify chromatin structure (Lorincz, Dickerson et al.
2004) and influence the elongation efficiency (Jacquier, 2009).
Resenly several works show that CGIs located far from known genes in intragenic regions
correspond to previously undetected promoters (Carninci et al., 2005; Medvedeva et al.,
2010) playing a role during development (Illingworth et al., 2011).
CTCF insulator protein forming a boundary of chromatin active regions (Bell & Felsenfeld,
2000) often binds CCCTC core motif common within CGIs.
CpG islands and mobile elements. There are a lot of repetitive sequences in human
genomes having high GC content, so many algorithms find CGI overlapping with repeats
(Alu-repeat in human (Graff et al., 1997) and B1-repeat in mouse (Yates et al., 1999)).
Cytosines within CGIs associated with Alu-repeats in normal cells are methylated, which in
turn represses the expansion of the repeat (Xing et al., 2004). Loss of methylation in Alu-
repeats is typical for tumor cells (Xie et al., 2010). Recently absence of methylation in Alu-
repeats was shown for germ line (Brohede & Rand, 2006). Ullu and Tschudi (Ullu &
Tschudi, 1984) believe that Alu-repeats are possessed pseudogenes of 7SL-RNA, and several
Alu families still contain inner promoter of RNA polymerase III (Britten et al., 1988). One
can expect that CGIs in Alu-repeats should have different DNA motifs comparing to CGIs in
promoters of protein-coding genes transcribed by PolII. Nevertheless, recent studies show
that pervasive PolII transcription is also a common feature for pseudogenes and transposons
(Frith et al., 2006).
Alu-repeats are source of spreading DNA methylation, so unmethylated CGIs contain TFBS
for Sp1 and other proteins to protect themselves from methylation (Caiafa & Zampieri,
2005). Recent studies show that Alu-repeats proximal to CpG islands could themselves form
a boundary protecting CpG islands from methylation (Feltus et al., 2003).
Taking into consideration all facts mentioned above, its obviously too early to exclude Alu-
and similar repeats out of attention speaking on CGIs functionality. Most of the authors (Takai
& Jones, 2002; H. Wu et al., 2010) try to build an algorithm for CGI search that avoid CGIs
around Alu-repeats. There are some differences in GC content, Obs/Exp
CpG
(Takai & Jones,
2002) or in cumulative mutual information of CpG dinucleotides (Su et al., 2009) between CGIs
found near Alu-repeats and around promoters of protein-coding genes. Yet most algorithms
excluded ab initio all repetitive sequences and therefore all of the CGIs located within them,
removing more than a half of CGIs in doing so. The question remains why the same sequences
in repetitive elements are of no use while in unique segments are essential.
CpG islands and replication origins. Sequence properties of replication origins in
mammals are not studied very well. There are some evidences that CpG islands near 3
region of the gene (Phi-van & Stratling, 1999) or in other genome regions can play a role of
replication origins (Rein et al., 1997; Rein et al., 1999), its important to know that some CpG
should be methylated in those regions for success of replication (Rein et al., 1999).
3.5 Approches for validation
Taking into consideration biological properties mentioned above, DNA methylation is a
logically relevant feature for CGI prediction validation. Complicated system of interactions
involving CGIs makes it obvious that considering CGI as merely unmethylated region is an
oversimplification. As far as DNA methylation plays important role in cell differentiation,
the same DNA region can be unmethylated in early stage of development and methylated in
later stages (reprogrammed DMR, rDMR), or unmethylated in one tissue and methylated in


458
another one (tissue-specific DMR, tDMR), or unmethylated in one allele and methylated in
another (allele-specific DMR, aDMR) as in case of imprinting or dosage compensation, or
demonstrate cross-individual differences in methylation (individual DMR, iDMR). More
appropriate way is to associate CGI with DMRs demonstrating absense (or decreased level)
of cytosine methylation only in one or few conditions.
Nevertheless even methylated CGIs play a role in transcription regulation, some of them
contains TSS of protein-coding (Shen et al., 2007) or non-coding genes (Medvedeva et al.,
2010). Recently a mechanism of transcription activation by binding of the C/EBP
transcription factor to the methylated CRE motif (TGACGTCA) was demonstated (Rishi et
al., 2010). Thus, the absence of methylation shouldnt be the only criterion for CGIs
verification.
Resently a lot of works dedicated to prediction of DNA methylation status in different
normal tissues ((Bock et al., 2008; Zhao & Han, 2009) and refs in them) and cancer (Feltus et
al., 2006) appeared. Various machine leaning techniques (support-vector machine (Bhasin et
al., 2005; Das et al., 2006), alternative decision trees (Carson et al., 2008), discriminant
analysis (Feltus et al., 2003)) were used to distinguish between methylated and
unmethylated regions. Authors use GC content, different di- and tri nucleotides (Das et al.,
2006; Fang et al., 2006), Alu-repeat location (Das et al., 2006; Fang et al., 2006), TpG fraction,
TFBS, repeats, predicted DNA structures (Bock et al., 2006) and other DNA patterns and
properties (Bhasin et al., 2005; Bock et al., 2007; Oakes et al., 2007; Carson et al., 2008; Ehrich
et al., 2008) as parameters for those studies. Results obtained by different authors are
incomparable, as in every case the model is built on distinct set of tissues and usually not in
a genome-wide manner. Features demonstrating high selectivity in one work dont do the
same in other works. The consistency of features is low, so one can conclude that those
models are overlearned.
Promoter proximity is another traditional key feature for CGI validation. The most popular
criterion is a fraction of predicted CGIs located near promoter regions of protein coding
genes. As a negative set Alu-repeats are usually used. SWM with higher thresholds for
length, GC content and Obs/Exp
CpG
(Takai & Jones, 2003; Han & Zhao, 2009) and clustering
algorithms (Glass et al., 2007; Hackenberg et al., 2010a; H. Wu et al., 2010) show best results.
Takai-Jones algorithm predicts 40% of CGIs to be located near promoters of RefSeq genes,
CpGcluster can reach the amount of 50% of all CGIs to be near promoter regions (with p-
value = 1.0e-20). Wu and colleagues (H. Wu et al., 2010) believe that CGHW predicts more
CGI to be located near promoters of RefSeq genes comparing to UCSC CGI and CG clusters.
Despite the fact that about half of CGIs are located near TSS of protein-coding genes the rest
are not. Lately various evidences of pervasive transcription appear (Carninci et al., 2005).
New high-throughput techniques (CAGE, SAGE, ets) identify at least ten times more
transcriptionally active regions comparing to number of protein-coding genes. Most of those
regions contain TSS for ncRNA of different types. CGIs located far from TSS of protein-
coding genes can act as their promoters. Nowadays discovery of new protein-coding genes
is rare occasion. Nevertheless our knowledge about ncRNA genes is extremely uncomplete.
On the other side, one shouldnt forget that mammalian genomes have not only CGI-
dependent promoters, but also TATA-dependent ones (Saxonov et al., 2006). The proportion
of both types is still unclear. Therefore fraction of CGIs associated with protein-coding genes
promoters is not an appropriate measure.
Other genomic features, like insulators, replication origins, recombination hot-spots, are also
co-located with CGIs and make the whole picture more complicated. Its also becoming clear


459
that CGI is not functionally equipotential throughout the length. CGI is not only a region
with high GC content and CpG frequency. Even in very early works on CGIs (G/C)-box was
mentioned as its structure element. Currently, its obvious that not only Sp1 but also a lot of
different TFs bind DNA within CGIs, so a huge fraction of them contains TFBS and their
clusters. Also, at least some CGIs have boundary regions containing binding sites for Sp1,
CTCF, VEZF1 or other TFs. Recently it was shown that G-quadruplex could also form a
boundary of CGIs. It should be emphasized that quality of biologically relevant feature
prediction is higher, if the method uses not only CGI prediction but includes other sequence
properties. Therefore the concept of complex CGI definition based not only on GC or CpG
content but also on other features like TFBS, repeats or DNA structure elements looks
promising.
4. Unsolved problems and perspectives
Despite the huge amount of works in the area commonly accepted definition of CpG islands
still doesnt exist. Most likely such situation is a result of difficulties with biological
verification of predictions (Segal, 2006). Authors of SWMs and to lower extend of clustering
algorithms choose the parameters arbitrarily complicating biological interpretations.
Authors of machine-learning techniques usually find too many distinguishing parameters
important in their models, which are not important in modeling of similar processes in other
cases.
Specifically it should be emphasized that all attempts to construct CGI prediction algorithm
based on simple DNA sequence properties (GC content, Obs/Exp
CpG
, distance between
neibouring CpG dinucleotides) having in mind prediction of complex biological feature
(promoter regions, unmethylated regions and so on) bring about a high level of false
positive predictions. For example, in case of promoter CGI prediction at least one third of
CGIs are located far from promoters. It admits of no doubt that existing CGI searchers find a
chimeric class of DNA segments, which dont have single common function. A collection of
DNA motifs relevant to different biological functions could result into more adequate CGI
definition. For instance, GC-skew and known core promoter elements could help to find
CGI or regions within them related to TSS.
Speaking on another feature of CGIs, namely lack of DNA methylation, it should be
mentioned that new high-throughput techniques show that not all CpG within CGIs are
unmethylated in normal cells, as previously believed. Nowadays it became clear that not
only CpGs but also CpNpGs are subject to methylation (Lister et al., 2009). Such a motif also
should be included in CGI prediction model (Hackenberg et al., 2010b).
The ability of a CGI searcher to predict DMRs but not unmethylated regions seems more
appropriate for quality evaluation. (Dai et al., 2008; Rakyan et al., 2008; Previti et al., 2009).
Unfortunately now we are still lack of high-quality and high-resolution data on genome-
wide DNA methylation in different tissues, states of developmet and conditions. High-
throughput techniques, like MeDIP, MeDIP-seq (Down et al., 2008), MethylCap-Seq
(Brinkman et al., 2010), bisulphyte conversion based methods (RRBS (Eckhardt et al., 2006)
and Methyl-seq (Lister et al., 2009)), let us hope for a complete map of DMRs in the nearest
future, which will help with CGI validation.
There is a lot of evidences that methylated cytosine also could play important functional role
as sites for methyl-binding proteins. We still havent enougth relaibale data on motif
preferences for all such proteins but we expect ChIP-seq (Mardis, 2007) technique to help


460
with the issue. There are proofs showing that its premature to exclude Alu- and other
repetitive mostly methylated sequences out of considereation speaking on CGI functions.
To resolve mentioned problems it is necessary to figure out as many biological functions
associating with CGIs as possible and to find out structure elements within CGI relating to
those functions or to separate CGI on several different functional groups. Such approach
should result in more precise and biologically adequate CGIs definition and, therefore
construction of relevant algorithm with low false positive and negative rates which in turn
will improve our knowledge in genetic and epigenetic regulation of genome functioning.
5. Comparison of different algorithms
A lot of comparisons between algorithms for CGI search have been performed. This work is
focused on study of various genome features potentially relates to CGIs. Three algorithms
for CpG islands search participate in the comparison: UCSC CGI, CpGcluster (with p-value
threshold of clusters equal to 1.0e-10, 1.0e-15, and 1.0e-20) and CGHW (the algorithm
implemented by Wu and colleagues). I prefer to focus on the algorithms of a new wave
and UCSC CGI as a reference because the last one is the most widespread now.
ENCODE regions of human genome (version hg18) were used for the study. All annotations
were downloaded from http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/.
Standard sensitivity (3) and specificity (4) measures for prediction quality were used.
Sn = L
TP
/ (L
FP
+L
FN
), (3)
Sp = L
TN
/ (L
FP
+L
TN
), (4)
where L
TP
total length (bp) of overlap of CGIs with tested annotation, L
FP
total length
(bp) of CGIs not overlapping with tested annotation, L
FN
- total length (bp) of tested
annotation not overlapping with CGIs, L
TN
- total length (bp) of ENCODE regions not
overlapping neither with tested annotation no with CGIs.
5.1 Basic statistics
As a first step I collected the summary of statictical properties of CGIs predicted by different
algorithms. CGI HW covers more then 2.2 % of total length of all ENCODE regions.
CpGcluster (p-value 1.0 e-20 as recommended in (Hackenberg et al., 2010a)) demonstrate the
smallest genome coverage of 0.6%. CpGcluster predicts shorter CGIs with higher average
GC-content and Obs/Exp
CpG
value comparing to other algorithms. UCSC CGI obtains the
largest average number of CpGs per one CGI.

UCSC CGI HW CpGcluster10 CpGcluster15 CpGcluster20
#CGI 507 1124 1093 633 418
CGI total length 396722 685514 303160 222603 172676
avarage length 782 610 277 352 413
avarage GC content 0.66 0.64 0.7 0.71 0.72
avarage #CpG per CGI 71 48 29 38 46
avarage Obs/ExpCpG 0.86 0.74 0.91 0.92 0.92
ENCODE fraction 0.0132 0.0229 0.0101 0.0074 0.0058
Table 1. Basic statistics for different CGIs.


461
In general one could see that CGI HW finds more relaxed CGIs comparing to UCSC CGI
(with lower GC-content, Obs/Exp
CpG
value and CpG frequency), whereas CpGcluster finds
more strict CGIs comparing to UCSC CGI.
5.2 Regulatory potential
TSS prediction. Its widely accepted that a large fraction of CGIs is found around TSS of
protein-coding genes. Recent studies show that total amount of TSS is about 10-times higher
than the amount of protein-coding genes, so it seems more appropriate to test the CGI
searchers for their ability to find TSS of any type. Several experimental techniques are able
to detect any type of TSSs. Cap analysis gene expression (CAGE) is one of the most known
techniques to produce a snapshot of the 5' ends of the total cellular RNA transcribed by
PolII. A collection of CAGE-tags (encodeRikenCagePlus and encodeRikenCageMinus tables
from UCSC) was used as a representative set of PolII TSS.

UCSC CGI CGI HW CpGcluster10 CpGcluster15 CpGcluster20
CGI fraction 0.0136 0.0090 0.0130 0.0152 0.0164
CAGE fraction 0.7274 0.7909 0.4632 0.4331 0.3903
Sn 0.0136 0.0091 0.0128 0.0149 0.0158
Sp 0.9869 0.9773 0.9900 0.9927 0.9943
Table 2. CAGE-tags clusters within different CGIs.
Table 2 shows that CGI HW has the lowest sensitivity, although they obtain the highest
fraction of CAGE-tags clusters. CpGcluster20 demonstrates the highest selectivity and
specificity but obtain only 39% of CAGE-tags clusters. UCSC CGI has the intermediate
values of Sn and Sp.
TFBS prediction. Although TFBS prediction is a classical problem for computational
molecular biology, prediction of one single but highly reliable TFBS still remains tricky. I
used TFBS conserved in the human/mouse/rat alignment based on Transfac Matrix
Database (tfbsConsSites and tfbsConsFactors tables from UCSC). Keeping in mind that
using of conserved TFBS leads to omission of all types of species-specific regulation regions,
conserved TFBS are more likely to be functional comparing to other predicted TFBS.
Table 3 demonstrates that CpGcluster predicts CGI with fewer different TFs and lower
sensitivity comparing to USCS CGI and CGI HW. The highest fraction of total TFBS length is
covered by CGI HW, the very same algorithm shows the highest sensitivity and the lowest
specificity. Its not obvious what fraction of the CGIs one should expect to be covered by
TFBS but CpGcluster20 demonstrates the largest coverage (about 19 %).

#TF 167 167 161 154 153
CGI fraction 0.1834 0.1347 0.1896 0.1915 0.1917
TFBS fraction 0.0860 0.1098 0.0688 0.0509 0.0393
Sn 0.0676 0.0696 0.0567 0.0443 0.0355
Sp 0.9889 0.9796 0.9916 0.9938 0.9952
Table 3. Conserved TFBS within different CGIs.


462
As its difficult to estimate the expected coverage of TFBS, I compared the coverage of CGIs
with the coverage of their adjacent regions of 100 bp. Results in Table 4 show that all
adjacent to CGI regions contain conserved TFBS.

#TF 157 167 166 162 151
CGI fraction 0.0564 0.0648 0.0871 0.0859 0.0820
TFBS fraction 0.0069 0.0177 0.0231 0.0132 0.0083
Sn 0.0063 0.0143 0.0189 0.0117 0.0077
Sp 0.9967 0.9928 0.9931 0.9960 0.9974
TFBS ratio 12.38 6.21 2.98 3.86 4.72
Table 4. Conserved TFBS within +/- 100 bp around different CGIs.
Last row of the Table 4 demonstrates the reduction of coverage in CGI adjacent regions
comparing to CGI bodies. The adjacent regions of UCSC CGI and CGI HW contain more
then 12 and 6 times less TFBS comparing to CGI body respectively. One should expect
some TFBS around CGI which can function as CGI's boundaries. One the other hand, if
we believe that CGI itself is the regulatory region, expected amount of TFBS in the
adjacent regions should be dramatically lower comparing to CGI body, which is not the
case for CpGcluster.
Insulators. CTCF is well known as a DNA binding protein acting both as transcriptional
factor and insulator protein. To test which CGI prediction algorithm finds more CTCF
binding sites I used data on CTCF binding (oregano and oreganoAttr tables from UCSC).
One can see that CGI HW shows the highest sensitivity in CTCF binding prediction. Its also
important to mention that CGIs from CGI HW contain more than 25% of all CTCF sites.
CpGcluster10 shows the second best result, and the quality of prediction decreases in case of
CpGcluster15 and CpGcluster20.

CGI fraction 0.0809 0.0658 0.0503 0.0569 0.0478
CTCF fraction 0.1395 0.2517 0.1871 0.1224 0.0680
Sn 0.0267 0.0434 0.0305 0.0241 0.0157
Sp 0.9872 0.9806 0.9916 0.9939 0.9953
Table 5. CTCF binding sites within different CGIs.
DNase sensitivity regions. DNase sensitivity regions are often considered as regions of
open chromatin which correspond to regulatory regions of all types. To test what algorithm
predicts CGI more often associated with DNase sensitivity regions I use joined data for
several tissues available in UCSC (table wgEncodeRegDnaseClustered). All CGIs
demonstrate rather good association with DNase sensitivity regions, at least one third of
their length is located in sensitive area. UCSC CGI shows highest sensitivity and rather good
spesifisity. Vast fraction of CpGcluster CGIs are also associated with DNase sensitivity
regions; althougth sensivity of the algorithm is not very good.


463
CGI fraction 0.6047 0.3221 0.4312 0.5872 0.6040
DNase fraction 0.0768 0.0707 0.0418 0.0418 0.0334
Sn 0.0789 0.0655 0.0413 0.0424 0.0338
Sp 0.9942 0.9827 0.9936 0.9966 0.9975
Table 6. DNase sensitivity regions within CGIs predicted by different algorithms and
quality of prediction.
Differently methylated regions. Data on regions differently methylated during
development was downloaded from the UCSC (table rdmr). Table 7 shows that CGI HW
predicts CGI located near over 43% of all rDMRs. This algorithm demonstrates also the best
sensitivity in this case. It shoud be menthioned that CpGcluster20 has the lowest sensitivity
and those CGIs are located near only 7% of rDMRs.

#rDMR fraction 0.2500 0.4310 0.2241 0.1293 0.0776
CGI fraction 0.0170 0.0262 0.0179 0.0161 0.0137
rDMR fraction 0.0534 0.1424 0.0432 0.0284 0.0187
Sn 0.0132 0.0231 0.0130 0.0105 0.0080
Sp 0.9869 0.9776 0.9900 0.9927 0.9943
Table 7. rDMRs within different CGIs.
Replication origins. To figure out if there is any preference for replication origins to be
found by one of CGI searchers data from encodeUvaDnaRepOriginsNSGM table were used.
Only CGI HW and CpGcluster10 find 5 and 2 replication origins within or around (+/- 100
bp) CGI respectively. Other algorithms (and CpGcluster with more strict parameters) are
unable to find any replication origins.
Polymorphic loci. Data from SNP130 were used for study of polymorphic loci within
different CGIs. CGI from CGI HW contains the highest fraction of SNPs and demonstrates
highest sensitivity, so one should expect more interindividual variants within those CGIs.

CGI fraction 0.0072 0.0082 0.0080 0.0073 0.0066
SNP fraction 0.0140 0.0276 0.0120 0.0080 0.0056
Sn 0.0048 0.0064 0.0049 0.0038 0.0031
Sp 0.9868 0.9771 0.9899 0.9926 0.9942
Table 8. SNPs within different CGIs.
6. Conclusions
In summary, no one algorithm for CGI search predicts all biologically relevant features
with appropriate accuracy. In all cases a lot of both false positives and false negatives
appear.


464
All algorithms participating in competition have its strong sides. CpGcluster (p-value = 1.0e-15
and p-value = 1.0e-20) demonstrate the highest specificity in TSS prediction. Although such
CGIs obtain the smallest fraction of CAGE-tags, this may be not a disadvantage as we dont
know for sure the proportion of GC- and AT-rich promoters. The largest fraction of CGIs
length is covered by TFBS in case of CGIs predicted by CpGcluster, on the other hand the
largest part of their adjacent regions is also covered by TFBS. This brought me to conclusion
that CpGcluster finds cropped promoter CGIs, espessially in case of p-value = 1.0e-20.
On the contrary CGI HW demonstrates the best sensitivity in CTCF binding sites and rDMR
prediction. CGI from CGI HW are associated with at least some of origins of repliacation,
thereas other algoritms (with recommended parameters) dont. They are also more prone to
find diversities between humans. Also those CGIs find the highest fraction of TSS. So, CGI
HW finds regions with broad regulatory potential. However all those features are related to
DNA methylation, which allow me to assume that CGI HW finds DMR-associated CGIs.
UCSC CGI demonstrates moderate behavior. This algorithm has intermediate sensitivity
both in TSS and rDMR prediction. Those CGIs have the highest decrease of TFBS in CGI
adjasent regions and the highest sensitivity to DNase. It looks like UCSC finds CGI around
promoter and also includes regulation regions, so those are promoter region CGIs.
Its quite clear that CGI is a complex object, which doesnt correspond to any single
biological feature. It seems more appropriate to segregate a class of interconnected
biological features: differential DNA methylation, active transcription at least in one cell
type or development stage and replication. CGI HW algorithm made the first step in this
direction, whereas CpGcluster (with high threshold for p-value) moves to the opposite
direction and finds specific narrow class of promoters. Traditional UCSC approach still
stands ground demonstrating comparable or in some points even higher quality. Hence the
CpG island problem is still far from final solution.
7. Acknowledgments
Author is very grateful to N. Oparina, V. Makeev, I. Artamonova and A. Favorov for fruitful
discussions on the topic of this article. This study was partially supported by RFBR grant 11-
04-02016-a and by the state contract P1376 of the Federal Special Program Scientific and
educational human resources of innovative Russia for 2009 2013.
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22
Translational Oncogenomics and Human
Cancer Interactomics: Advanced Techniques
and Complex System Dynamic Approaches
I. C. Baianu
AFC-NMR & NIR Microspectroscopy Facility,
College of ACES, FSHN & NPRE Departments,
University of Illinois at Urbana, Urbana, IL
USA
1. Introduction
An overview of translational, human oncogenomics, transcriptomics and cancer
interactomic networks is presented together with basic concepts and potential, new
applications to Oncology and Integrative Cancer Biology. Novel translational oncogenomics
research is rapidly expanding through the application of advanced technology, research
findings and computational tools/models to both pharmaceutical and clinical problems. A
self-contained presentation is adopted that covers both fundamental concepts and the most
recent biomedical, as well as clinical, applications. Sample analyses in recent clinical studies
have shown that gene expression data can be employed to distinguish between tumor types
as well as to predict outcomes. Potentially important applications of such results are
individualized human cancer therapies or, in general, personalized medicine. Several cancer
detection techniques are currently under development both in the direction of improved
detection sensitivity and increased time resolution of cellular events, with the limits of
single molecule detection and picosecond time resolution already reached. The urgency for
the complete mapping of a human cancer interactome with the help of such novel, high-
efficiency, low-cost and ultra-sensitive techniques is also pointed out.
1.1 Current status in translational genomics and interactome networks
Upon completion of the maps for several genomes, including the human genome, there are
several major post-genomic tasks lying ahead such as the translation of the mapped
genomes and the correct interpretation of huge amounts of data that are being rapidly
generated, or the important task of applying these fundamental results to derive major
benefits in various medical and agricultural biotechnology areas. Translational genomics is
at the center of these tasks that are running from transcription through translation to
proteomics and interactomics. The transcriptome is defined as the set of all transcripts or
messenger RNA (mRNA) molecules produced through transcription from DNA sequences
by a single cell or a cell population. This concept is also extended to a multi-cellular
organism as the set of all its transcripts. The transcriptome thus reflects the active part of the
genome at a given instant of time. Transcriptomics involves the determination of mRNAs


474
expression level in a selected cell population. For example, an improved understanding of cell
differentiation involves the determination of the stem cell transcriptome; understanding
carcinogenesis requires the comparison between the transcriptomes of cancer cells and
untransformed (normal) cells. However, because the levels of mRNA are not directly
proportional to the expression levels of the proteins they are encoding, the protein
complement of a cell or a multi-cellular organism needs to be determined by other techniques,
or combination of techniques; the complete protein complement of a cell or organism is
defined as the proteome. When the network (or networks) of complex protein-protein
interactions (PPIs) in a cell or organism is (are) reconstructed, the result is called an interactome.
This complete network of PPIs is now thought to form the backbone of the signaling
pathways, metabolic pathways and cellular processes that are required for all key cell
functions and, therefore, cell survival. Such a complete knowledge of cellular pathways and
processes in the cell is essential for understanding how many diseases -- such as cancer (and
also ageing) originate and progress through mutation or alteration of individual pathway
components. Furthermore, determining human cancer cell interactomes of therapy-resistant
tumors will undoubtedly allow for rational clinical trials and save patients lives through
individualized cancer therapy. Since the global gene expression studies of DeRisi et al in 1997,
translational genomics is very rapidly advancing through the detection in parallel of mRNA
levels for large numbers of molecules, as well as through progress made with miniaturization
and high density synthesis of nucleic acids on microarray solid supports. Gene expression
studies with microarrays permit an integrated approach to biology in terms of network
biodynamics, signaling pathways, protein-protein interactions, and ultimately, the cell
interactome. An important emerging principle of gene expression is the temporally coordinated
regulation of genes as an extremely efficient mechanism (Wen et al 1998) required for complex
processes in which all the components of multi-subunit complexes must be present/available
in defined ratios at the same time whenever such complexes are needed by the cell. The gene
expression profile can be thought of either as a signature/ fingerprint or as a molecular
definition of the cell in a specified state (Young, 2000). Cellular phenotypes can then be inferred
from such gene expression profiles. Success has been achieved in several projects that profile a
large number of biological samples and then utilize pattern matching to predict the function of
either new drug targets or previously uncharacterized genes; this compendium approach has
been demonstrated in yeast (Gray et al 1998; Hughes et al 2000), and has also been applied in
databases integrating gene expression data from pharmacologically characterized human
cancer lines (NCI60, http://dtp.nci.nih.gov), or to classify cell lines in relation to their tissue of
origin and predict their drug resistance or chemosensitivity (Weinstein et al, 1997; Ross et al
2000, Staunton et al 2001). Furthermore, sample analyses in clinical studies have shown that
gene expression data can be employed to distinguish between tumor types as well as to
predict outcomes (Golub et al 1999; Bittner et al, 2000; Shipp et al 2002; Furteal et al., 2004). The
latter approach seems to lead to important applications such as individualized cancer therapy
and personalised medicine. On the other hand, such approaches are complemented by
studies of protein-protein interactions in the area called proteomics, preferably under
physiological conditions, or more generally still, in cell interactomics. Several technologies in
this area are still developing both in the direction of improved detection sensitivity and time
resolution of cellular events, with the limits of single molecule detection and picosecond time
resolution already attained. In order to enable the development of new applications such
techniques will be briefly described in the next section, together with relevant examples of
their recent applications.

Oncogenomics and Cancer Interactomics

475
1.2 Basic concepts in transcription, translation and interactome networks
The analysis of bionetwork dynamics of protein synthesis considered as a channel of
information operates through the formation of protein amino acid sequences of
polypeptides via translation of the corresponding polynucleotide sequences of (usually
single stranded, messenger) ribonucleic acid, that is:
DNA (gene) transcription mRNA translation into a polypeptides aminoacid sequence
protein (quaternary) assembly from polypeptide subunits.
Although not shown in this scheme, several key enzymes make such processes both efficient
and precise through highly-selective catalysis; moreover, the protein assembly involves both
specific enzymes and ribosome assembly lines. Furthermore, such processes are
compartmented in the mammalian cells by selective intracellular membranes; this seems to
be also important for cell cycling and the control of cell division. On the other hand, the
reverse transcription, RNA DNA, does also occur (under certain conditions), catalized by a
reverse transcriptase that contains both polypeptide chains and an RNA (master) strand. If
error free, the first of these two sequence of processes which are of fundamental biological
importance-- generates true replicas of the information contained in the sense codons of the
genes that are transcribed into mRNA anti-codons. Recall also that DNA stores information
in the neucleotide bases A (Adenine), C (Cytosin), G (Guanine) and T (Thymine), and that a
triplet of such nucleotides in the DNA sequence is called a codon, which may encode
unambiguously just the information necessary to specify a single amino acid. Moreover, the
genetic code is quasi-universal, and capable of reverse transcription' from certain types of
RNA back into DNA. Notably also, not all nucleotide or codon sequences present in the
genome (DNA) are transcribed in vivo. Typically only a small percentage is transcribed. The
transcribed (mRNA) sequences form what is naturally called the transcriptome; the protein--
encoded version of the transcriptome is called the proteome, and upon including all protein--
protein interactions for various cellular states one obtains the (global) interactome network.
More generally, biological interactive networks as a class of complex bionetworks consist of
local cellular communities (or organismic sets') that are organized and managed by their
characteristic selection procedures. Thus, in any partitioning of the organismal, or cell,
structure, it is often necessary to regulate the local properties of the organism rather than the
global mechanism, which explains an organism's need for specialized, modular
constructions'. Such a modular, complex system biology approach to modeling signaling
pathways and modifications of cell-cycling regulatory mechanisms in cancer cells was
recently reported (Baianu, 2004); several consequences of this approach were also
considered for the proteome and interactome networks in a prototype cancer cell model
(Prisecaru and Baianu, 2005). Note, on the other hand, that there seem to be also present in
the living cell certain proteins and enzymes that are involved in global intra-cellular
interactions which are thought to be essential to the cell survival and cells flexible adaptation
to stresses or challenge. Recent modeling techniques draw from a variety of mathematical
sources, such as: topology (including graph theory), biostatistics, stochastic differential
equations, Boolean networks, and qualitative system dynamics (Baianu, 1971a; de Jong et al
2000; 2003, 2004). Non--boolean network models of genetic networks and the interactome
were also developed and compared with the results of Boolean ones (Baianu, 1977, 1984, 1987;
Georgescu, 2006; Baianu, 2005; Baianu et al. 2006). The traditional use of comparatively rigid
Boolean networks (reviewed extensively, for example in Baianu, 1987) can be thus extended
through flexible, multi--valued (non--Boolean) logic algebra bionetworks with complex, non-
linear dynamic behaviors that mimic complex systems biology (Rosen, 2000).


476
The results obtained with such non--random genetic network models have several important
consequences for understanding the operation of cellular networks and the formation,
transformation and growth of neoplastic network structures. Non--boolean models can also
be extended to include epigenetic controls discussed in Section 6, as well as to mimic the
coupling of the genome to the rest of the cell through specific signaling pathways that are
involved in the modulation of both translation and transcription control processes. The latter
may also provide novel approaches to cancer studies and, indeed, to developing
individualized cancer therapy strategies and novel anti-cancer medicines targeted at specific
signaling pathways involved in malignant tumors resistance to other therapies.
2. Techniques and application examples
2.1 DNA microarrays
DNA microarray technology is widely employed to monitor in a single experiment the gene
expression levels of all genes of a cell or an organism. This includes the identification of
genes that are expressed in different cell types as well as the changes in gene expression
levels caused, for example, by differentiation or disease. The terabytes of data thus obtained
can provide valuable clues about the interactions among genes and also about the
interaction networks of gene products. It has been reported that cDNA arrays were
pioneered by the Brown Laboratory at Stanford University (Brown and Botstein, 1999; URL:
http://cmgm.stanford.edu/pbrown/mguide/index.html). Several quantitative and high-
density DNA array applications were then reported in rapid succession (Schena et al 1995;
Chee et al 1996; Brown and Botstein, 1999). Such microarrays are generated by automatically
printing double-stranded cDNA onto a solid support that may be either glass silicon or
nylon. The essential technologies involved are robotics and devlopment/selection of
sequence-verified and array-formatted cDNA clones. The latter ensures that both the
location and the identity of each cDNA on the array is known. Sequence-verified and array-
formatted cDNA clone sets are now available from companies such as Incyte Genomics
(Palo Alto, CA; URL: http://www.synteni.com/) and Research Genetics (Huntsville, AL;
URL: http://www.resgen.com/). In cDNA-based gene expression profiling experiments,
the total RNA is extracted from the selected experimental samples and the RNA is
fluorescently labeled with either cye3- or cye5-dUTP in a single round of reverse
transcription. The latter have several advantages: they are readily incorporated into cDNA
by reverse transcription, they exhibit widely separated excitation and emission spectra, and
also they possess good photostability. Such fluorescently--labeled cDNA probes are then
hybridized to a single array through a competitive hybridization reaction. Detection of
hybridized probes is achieved by laser excitation of the individual fluorescent markers,
followed by scanning using a confocal scanning laser microscope. The raw data obtained
with a laser scanning systems is represented as a normalized ratio of cye3: cye5 and
automatically color coded; thus, red color is conventionally selected to represent those genes
that are transcriptionally upregulated in the test versus the reference, whereas green color
represents genes that are downregulated; those genes that exhibit no difference between test
and reference samples are shown in yellow. The analysis of the gene expression data
obtained by such a high throughput microarray technology is quite complex and requires
advanced computational/bioinformatics tools as already discussed in Section 1.2. Other
aspects related to interactomics are discussed in Section 3. An alternative technology to
cDNA microarrays is discussed in the next section.


477
2.2 Oligonucleotide arrays
By combining oligonucleotide synthesis with photolithography it was possible to synthesize
specific oligonucleotides with a selected orientation onto the solid surface of glass or silicon
chips (Lockhart et al 1996; Wodicka L, et al 1997), thus forming oligonucleotides arrays. The
expression monitoring was then carried out by hybridization to high-density
oligonucleotide arrays (Lockhart et al 1996; Wodicka L, et al 1997). Commercially available
oligonucleotides array products include human, mouse and several other organisms. Each
gene included on the oligonucleotides array is represented by up to 20 different
oligonucleotides that span the entire length of the coding region of that gene. To reduce
substantially the rate of false positives, each of these oligonucleotides is paired with a
second mismatch oligonucleotide in which the central base in the sequence has been
replaced by a different base. As in the cDNA approach, fluorescently labeled probes are
generated from test and reference samples in order to carry out comparative gene
expression profiling. After cDNA amplification, the differential fluorescent signal is
detected with a laser scanning system and provides a map of the alterations in the
transcriptional profile between the test and reference samples that are being compared.
Dynamic analysis and further sophistication is added to such oligonucleotides array
capabilities by the techniques briefly discussed in Section 2.6. The molecular classification of
cancers is of immediate importance to both cancer diagnosis and therapy. Tumors with
similar histologic appearance quite often have markedly different clinical response to
therapy. Such variability is a reflection of the underlying cell line and molecular
heterogeneity of almost any tumor. Gene expression profiling has been successfully
employed for molecular classification of cancers. It would seem from available data that
each patient has her/his own molecular identity signature or fingerprint (Mohr et al 2002).
Thus, Ross et al. (2000) reported the gene expression analysis in 60 cancer cell lines utilized
in the Developmental Therapeutics Program by the National Cancer Institute (NCI) at NIH
(Bethesda, MD, USA); the report also stated that cell lines could be grouped together
according with the organ type and specific expression profiles corresponded to clusters of
genes. Similar findings were reported for ovarian and breast cancers; in the latter case, Perou
et al. (2000) reported that specific epithelial cell line genes clustered together and are
relevant in breast cancer subdivision into the basal- like and luminal groups. On the other
hand, the eventual use of microarray technologies for clinical applications will involve the
utilization of proteome and tissue arrays in addition to gene expression profiling by cDNA
microarrays and oligonucleotides arrays. Thus, tissue markers revealed unexpected
relationships, as in the case of gene expression analysis of small-cell lung carcinoma,
pulmonary carcinoid tissue and bronchial epithelial tissue culture (Anbazhagan et al 1999).
Because a single biomarker has serious limitations for clinical applications there is a need for
a battery of disease biomarkers that would provide a much more accurate classification of
cancers. High-density screening with microarray technologies is therefore valuable in
pharmacogenomic (individualized therapy), toxicogenomic, as well as in clinical -
diagnostic investigations.
2.3 Proteome arrays
In a manner similar to the transcriptome, the proteome does undergo both qualitative and
quantitative changes during pathogenesis, and this is also true in carcinogenesis. Proteome
array-based methodologies involve either proteins or protein-binding particles (DNA,
RNAs, antibody, or other ligands). Utilizing such proteome arrays one can respectively


478
study either differential protein expression profiling or protein-ligand interaction screening
under specified, or selected, physiopathological conditions. According to Kodadek (2001),
these two classes of practical applications of proteome arrays are respectively defined as
protein function and protein-detecting arrays. A protein-detecting array may consist of an
arrayed set of protein ligands that are employed to profile gene expression and therefore
make visible proteosignatures characterizing a selected cellular state or phase. In view of
the potential clinical importance of a proteomic survey of cancers, the hunt is now on for
such proteosignatures of cancer cells but the amount of data reported to date is still quite
limited. Already, the coupling of proteome arrays with high-resolution chromatography
techniques followed by mass spectrometry has provided powerful analytical tools with
which one can profile the protein expression in cancer cells. For example, a ProteinChip
TM

(Ciphergen Inc, Fremont, CA, USA) was successfully utilized to investigate the proteome of
prostate, ovarian, head and neck cancer cells (von Eggeling et al 2000). Such methods
identified protein fingerprints from which cancer biomarkers can also be obtained. A
reverse proteome array was also reported in which many extracted proteins from a patient
sample are printed onto a flat, solid support (Paweletz et al 2001); this reverse system was
then utilized to carry out a biochemical screening investigation of the signaling pathways in
prostate cancer. Through such investigations it was found that the carcinoma progression
was positively correlated with the phosphorylation state of Akt and negatively correlated
with ERK pathways; furthermore, the carcinoma progression was positively correlated with
the suppression of the apoptotic pathways, a finding which is consistent with the more
detailed, recent reports on cyclin CDK2 and transcriptional factors affected by CDK2 that
will be discussed in Section 4. Immunophentotyping of leukemias with antibody
microarrays was also reported (Belov, de la Vega, dos Remedios, et al 2001), and does
provide an increased antigen differentiation (CD) in leukemia processing.
2.4 Tissue arrays
The logical step after the identification of potential cancer markers through genomic and/or
proteomic array analysis is the evaluation of such cancer markers by tissue arrays/ tissue
chips for diagnostic, prognostic, toxicogenomic and therapeutic relevance. Such tissue
microarrays (TMAs) were often designed to contain up to 1000 sections of 5micron thick
sections, usually chemically--fixed and arrayed upon a glass slide. TMAs allow large-scale
screening of tissue specimens and can be utilized, for example, for the pathological
evaluation of molecular irreversible changes that are important for cancer research and
treatment. Therefore, they can speed up the process of translating experimental, or
fundamental, discoveries into clinical practice and improved cancer treatments.
TMAs have been utilized in cancer research in conjunction with fluorescence in situ
hybridization (FISH), to analyze in parallel the gene amplification in multiple tissue
sections thus allowing the researchers to map the distribution of gene amplification
throughout an entire tumor. This also allowed the monitoring of changes in gene
amplification during the cancer progression (Bubendorf et al 1999). Furthermore, utilizing
immunohistochemical staining of tissue arrays it was possible to measure the protein levels
in tumor specimens. Thus, topoisomerase II alpha was reported to be highly expressed in
patients with the poorest prognosis in oligodendrogliomas (Miettinen et al 2000). TMAs
may become a clinical validation, as well as a global tool; thus, recent studies reported this
technique to be highly efficient for the identification of molecular (irreversible) alterations


479
during cancer initiation and progression (Lassus et al 2001). A pathologist might, however,
object that the tissue microarray provides only a partial analysis of the tumor. The array-
based technologies briefly described here provide powerful means for functional analyses of
cancer and other complex diseases. Undoubtedly, much more can, and will be, done with
proteome or tissue arrays combined with other state-of-the-science spectroscopic techniques
as suggested in the following Sections 2.5, 2.6, 4 and 6.2.
The following three Sections 2.5 and 2.6 and 6.2 will illustrate how advanced, ultra-fast and
super-sensitive techniques can be used in conjunction with either nucleic acids or proteome
arrays to both speed up thousand-fold the microarray data collection (for nucleic acids,
proteins, ligand-binding, etc.) and also increase sensitivity to its possible limit--that of single
molecule detection.
2.5 Fluorescence correlation spectroscopy and fluorescence crosscorrelation
spectroscopy: applications to DNA hybridization, PCR and DNA binding
In the bioanalytical and biochemical sciences Fluorescence Correlation Spectroscopy (FCS)
techniques can be utilized to determine various thermodynamic and kinetic properties, such
as association and dissociation constants of intermolecular reactions in solution (Thompson,
1991; Schwille, Bieschke and Oehlenschlger, 1997). Examples of this are specific
hybridization and renaturation processes between complementary DNA or RNA strands, as
well as antigene-antibody or receptor-ligand recognition. Although of significant functional
relevance in biochemical systems, the hybridization mechanism of short oligonucleotide
DNA primers to a native RNA target sequence could not be investigated in detail prior to
the FCS/FCCS application to these problems. Most published models agree that the process
can be divided into two steps: a reversible first initiating step, where few base pairs are
formed, and a second irreversible phase described as a rapid zippering of the entire
sequence. By competing with the internal binding mechanisms of the target molecule such
as secondary structure formation, the rate-determining initial step is of crucial relevance for
the entire binding process. Increased accessibility of binding sites, attributable to single-
stranded open regions of the RNA structure at loops and bulges, can be quantified using
kinetic measurements (Schwille, Oehlenschlger and Walter, 1996).
The measurement principle for nearly all FCS/FCCS applications is based so far upon the
change in diffusion characteristics when a small labeled reaction partner (eg, a short nucleic
acid probe) associates with a larger, unlabeled one (target DNA/RNA). The average diffusion
time of the labeled molecules through the illuminated focal volume element is inversely
related to the diffusion coefficient, and increases during the association process. By calibrating
the diffusion characteristics of free and bound fluorescent partner, the binding fraction can be
easily evaluated from the correlation curve for any time of the reaction. This principle has been
employed to investigate and compare the hybridization efficiency of six labeled DNA
oligonucleotides with different binding sites to an RNA target in a native secondary structure
(Schwille, Oehlenschlger and Walter, 1996). Hybridization kinetics was examined by binding
six fluorescently labeled oligonucleotide probes of different sequence, length and binding sites
to a 101-nucleotide-long native RNA target sequence with a known secondary structure. The
hybridization kinetics was monitored and quantified by FCS, in order to investigate the overall
reaction mechanism. At the measurement temperature of 40C the probes are mostly
denatured, whereas the target retains its native structure. The binding process could be
directly monitored through diffusional FCS analysis, via the change in translational diffusion
time of the labeled 17-mer to 37-mer oligonucleotide probes HS1 to HS6 upon specific


480
hybridization with the larger RNA target. The characteristic diffusion time through the laser-
illuminated focal spot of the 0.5 m-diameter objective increased from 0.13 to 0.20 ms for the
free probe, and from 0.37 to 0.50 ms for the bound probe within 60 min. The increase in
diffusion time from measurement to measurement over the 60 min could be followed on a PC
monitor and varied strongly from probe to probe. HS6 showed the fastest association, while
the reaction of HS2 could not be detected at all for the first 60 min. Thus, FCS diffusional
analysis provides an easy and comparably fast determination of the hybridization time course
of reactions between complementary DNA/RNA strands in the concentration range from 10
-10

to 10
-8
M. The FCS-based methodology also permits rapid screening for suitable anti-sense
nucleic acids directed against important targets like HIV-1 RNA with low consumption of
probes and target. Because of the high sensitivity of FCS detection, the same principle can be
exploited to simplify the diagnostics for extremely low concentrations of infectious agents like
bacterial or viral DNA/RNA. By combining confocal FCS with biochemical amplification
reactions like PCR or 3SR, the detection threshold of infectious RNA in human sera could be
dropped to concentrations of 10
-18
M (Walter, Schwille and Eigen, 1996; Oehlenschlger,
Schwille and Eigen, 1996). The method allows for simple quantification of initial infectious
units in the observed samples. The isothermal Nucleic Acid Sequence-Based Amplification
(NASBA) technique enables the detection of HIV-1 RNA in human blood-plasma (Winkler,
Bieschke and Schwille, 1997). The threshold of detection is presently down to 100 initial RNA
molecules per milliliter by amplifying a short sequence of the RNA template (Schwille,
Oehlenschlger and Walter, 1997). The NASBA method was combined with FCS, thus
allowing the online detection of the HIV-1 RNA molecules amplified by NASBA
(Oehlenschlger, Schwille and Eigen, 1996). The combination of FCS with the NASBA reaction
was performed by introducing a fluorescently labeled DNA probe into the NASBA reaction
mixture at nanomolar concentrations, hybridizing to a distinct sequence of the amplified RNA
molecule. After having reached a critical concentration on the order of 0.1 to 1.0 nM (the
threshold for single-photon excitation / FCS detection is ~0.1 nm), the number of amplified
RNA molecules could be determined as the reaction continued its course. Evaluation of the
hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration
present at the beginning of amplification. The value of the initial HIV-1 RNA number enables
discrimination between positive and false-positive samples (caused, for instance, by carryover
contamination). This possibility of sharp discrimination is essential for all diagnostic methods
using amplification systems (PCR as well as NASBA). The quantification of HIV-1 RNA in
plasma by combining NASBA with FCS may be useful in assessing the efficacy of anti-HIV
agents, especially in the early infection stage when standard ELISA antibody tests often
display negative results. Furthermore, the combination of NASBA with FCS is not restricted
only to the detection of HIV-1 RNA in plasma.
On the one hand, the diagnosis of Hepatitis (both B and C) remains much more challenging.
On the other hand, the number of HIV, or HBV, infected subjects worldwide is increasing at
an alarming rate, with up to 20% of the population in parts of Africa and Asia being infected
with HBV. In contrast to HIV, HBV infection is not particularly restricted to the high-risk
groups.
Multi-photon (MPE) NIR excitation of fluorophores--attached as labels to biopolymers like
proteins and nucleic acids, or bound at specific biomembrane sites-- is one of the most
attractive options in biological applications of FCS. Many of the serious problems
encountered in spectroscopic measurements of living tissue, such as photodamage, light
scattering and auto-fluorescence, can be reduced or even eliminated. FCS can therefore


481
provide accurate in vivo and in vitro measurements of diffusion rates, mobility parameters,
molecular concentrations, chemical kinetics, aggregation processes, labeled nucleic acid
hybridization kinetics and fluorescence photophysics/ photochemistry. Several
photophysical properties of fluorophores that are required for quantitative analysis of FCS
in tissues have already been widely reported. Molecular mobilities can be measured by
FCS over a wide range of characteristic time constants from ~10
-3
to 10
3
ms.
Novel, two-photon NIR excitation fluorescence correlation spectroscopy tests and preliminary
results were obtained for concentrated suspensions of live cells and membranes (Baianu et al,
2007). Especially promising are further developments employing multi-photon NIR excitation
that could lead, for example, to the reliable detection of cancers using NIR-excited
fluorescence. Other related developments are the applications of Fluorescence Cross-
Correlation Spectroscopy (FCCS) detection to monitoring DNA- telomerase interactions, DNA
hybridization kinetics, ligand-receptor interactions and HIV-HBV testing. Very detailed,
automated chemical analyses of biomolecules in cell cultures are now also becoming possible
by FT-NIR spectroscopy of single cells, both in vitro and in vivo. Such rapid analyses have
potentially important applications in cancer research, pharmacology and clinical diagnosis.
2.6 Near infrared microspectroscopy, fluorescence microspectroscopy and infrared
chemical imaging of single cells
Novel methodologies are currently being evaluated for the chemical analysis of embryos
and single cells by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared
(FT-NIR) Microspectroscopy, Fluorescence Microspectroscopy. The first FT-NIR chemical
images of biological systems approaching 1micron (1m) resolution were reported (Baianu,
2004; Baianu et al 2004), and FT-NIR spectra of oil and proteins were obtained under
physiological conditions for volumes as small as 2m
3
. Related, HR-NMR analyses of oil
contents in somatic embryos were presented with nanoliter precision. Therefore,
developmental changes may be monitored by FT-NIR with a precision approaching the
picogram level when adequately calibrated by a suitable primary analytical method.
Indeed, detailed chemical analyses are now becoming possible by FT-NIR Chemical
Imaging and Microspectroscopy of single cells. The cost, speed and analytical requirements
are fully satisfied by FT-NIR spectroscopy and microspectroscopy for a wide range of
biological specimens. These techniques were also suggested to be potentially important in
functional genomics and proteomics research (Baianu et al 2004) through the rapid and
accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed
femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of
single molecule detection (SMD). Thus, microspectroscopic techniques allow for most sensitive
and reliable quantitative analyses to be carried out both in vitro and in vivo. In particular, MP
NIR excitation in FCS allows not only single molecule detection, but also non-invasive
monitoring of molecular dynamics and the acquisition of high-resolution, submicron imaging
of femtoliter volumes inside functional cells and tissues. Such ultra-sensitive and rapid NIR-
FCS analyses have therefore numerous potential applications in biomedical research areas,
clinical diagnosis of viral diseases, cancers and also in cancer therapy.
3. Mapping interactome networks
Mapping protein-protein interaction networks, or charting the global interaction maps, that
correspond through translation to entire genomes is undoubtedly useful for understanding


482
cellular functions, especially when such databases can be integrated into a wide collection of
biologically relevant data. A prerequisite for any ab initio determination of a selected
protein interactome network is to clone the open reading frames (ORFs) that encode each
protein present in the selected network. Note, however, that all current analyses involve the
assumption of a model together with some hidden, or implicit, assumptions about sampling,
noise levels, or uniformity/ accuracy in the database, and therefore, the ab initio claim is
subject to the restrictions imposed by such additional assumptions. More than 20,000 of
publicly accessible, full ORF clones have been already collected for human and mouse
protein-coding genes in the Mammalian Genome Collection (MGC; http://mgc.nci.nih.gov).
This community resource enables the next stages of human interactome analysis that will be
directed at obtaining a reliable map of the entire human protein interactome. An additional,
12,500 ORFs are now available from the Dana Farber Cancer Institute in Boston (USA) from
high-throughput, yeast two-hybrid (Y2H) analyses. A disconcerting aspect of the latest
human (partial) interactome studies by different methods is the little apparent overlap of the
new human interaction datasets with each other and/or with previously reported data. This
aspect will be further addressed later in this section; the principal cause for the lack of
overlap is likely to be caused by the low (<20%) overall coverage of the protein-protein
interactions selected in such studies. A possible solution to this problem has been suggested
(Warner et al 2006): several groups cooperating to produce networks of networks,
constructed from separatebut coordinatedinteraction mapping projects, each of which
would target a specific functionality related subset of proteins and interactions. A more
effective solution would be, however, to increase the throughput, accuracy and reliability of
PPI data through improved technologies (such as FCCS, or other techniques already
proposed in Section 2.5, for example), reduce significantly the cost of such analyses, as well
as improve the models employed for data analysis. Examples of improved modeling tools
for this purpose, such as logical, ontological genetics and categorical ones, that are also
appropriate for assembling the networks of networks as in the previous approach
suggested by Warner et al. (2006), were presented above in Section 1, and are described in
further detail in a recent report (Baianu et al 2006) and also in two forthcoming publications
(Baianu and Poli, 2011; Baianu et al 2010). Interactome network studies are currently
undertaken by a number of international research teams in the US, Europe and Japan
(CSH/WT, 2006; Warner et al 2006). These studies are currently undertaken only for
interactome subnetworks because of both technique and funding limitations. The organisms
studied were: yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans), fruitfly
(Drosophila melanogaster) and humans. Proteome networks were investigated for several,
specific, biological processes such as: DNA degradation, ubiquitin conjugation,
multivesicular formation, intracellular membrane traffick, signal transduction/ TNF
tumor necrosis and NF B mediated pathways, and early stages of T-cell signaling (for a
brief summary note the recent review by Warner et al 2006, and references cited therein).
Such challenging studies face both methodological problems such as limited sampling (Han
et al 2006) and consideration of only pairwise (binary) protein-protein interactions, and also
the more serious technical problem of false-positive interactions in the presence of a
significant noise levels associated with the experimental technologies and design currently
employed in such studies. Such limitations should be borne in mind (Han et al 2006) when
global topology predictions are made for the whole interactome based on partial,
incomplete data obtained for subnetworks that may contain less than 20% of the entire
interactome network. On a more optimistic note are the recent attempts at comparing the


483
cancer protein, human interactome (sub) networks with normal human interactome
networks that involve multiple protein-protein interactions (Jonsson and Bates, 2006). The
latter studies reduced the noise level in the human protein interaction data by employing
an orthology-based method described previously by Jonsson et al. (2006). This method
claims to reduce the noise level in protein-interaction (PPI) data by identifying putative
interactions based on homology to experimentally determined interactions in a range of
different species; both the DIP (Salwinsky et al 2004) and the MIPS, Mammalian Protein
Protein Interaction (Pagel et al 2005) databases were utilized. Furthermore, the complete
interactome data set that was employed is available as Supplementary Material from loc. cit.
The conclusions was drawn that cancer proteins have an increased frequency of protein-
protein interactions in comparison with the proteins that were studied in normal cells, and
this was interpreted as evidence indicating an underlying evolutionary pressure to which cancer
genes, as genes of central importance are subjected. It remains to be seen, however, if human
interactome studies-- which occur with increasing frequency-- have indeed overcome the
sampling objections raised by Han et al. (2006). The more extensive interactome data and
analysisthough still quite limited- that has been reported to date is readily available and
includes the following: Y2H (partial data-based) interactome maps for C. elegans (Li et al
2004) and Drosophila melanogaster (Giot et al 2003; Formstecher et al 2005), and also proteome
maps obtained by co-affinity purification followed by mass spectrometry analysis in yeast-
Saccharomyces cerevisiae (co-AP/MS: Gavin et al 2002; Ho et al 2002; Han et al 2004). The
reports on the microbial transcriptional regulation network of Escherichia coli (Shen-Orr et al
2002) and on Helicobacter pylori protein complexes in the proteome map (Terradot et al 2004)
are also worthile mentioning in this context. A first-draft of the human interactome has also
been reported (Lehner and Fraser, 2004); although this human interactome map does not
seem to have been included in the computational investigations of Han et al. (2006), it
remains to be verified, or validated, by further extensive studies with improved technology
and adequate models for a more comprehensive data analysis. The comprehensive two-
hybrid analysis for exploring the protein interactome network was previously reported by
Ito et al. (2001). Alternative interaction mapping strategies have also been developed over
the last five years. An example is the tandem affinity purification (TAP) in conjunction with
liquid chromatography tandem mass spectrometry (LC-MS/MS; see, for example, Gavin et
al 2006). Such methods have, however, both advantages and limitations. An interesting, new
approach to the determination of protein complexes has been developed that involves a
combination of fluorescence spectroscopy with peptide microarrays (Stoevesandt, cited in
Warner 2006); this methodology was then applied to investigate T-cell signaling.
4. Cell cyclins expression and modular cancer interactome networks
Carcinogenesis is a complex process that involves dynamically inter-connected biomolecules
in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments that form
numerous inter-related pathways referred to as networks. One such family of pathways
contains the cell cyclins. Cyclins are often overexpressed in cancerous cells (Dobashi et al
2004).
Our novel theoretical analysis based on recently published studies of cyclin signaling, with
special emphasis placed on the roles of cyclins D1 and E, suggests novel clinical trials and
rational therapies of cancer through re-establishment of cell cycling inhibition in metastatic
cancer cells.


484
4.1 Cyclins
Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/division
components, including the tumor suppressor gene TP53 and its product, the Thomsen-
Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax), which all play major
roles in carcinogenesis of many cancers. Cyclin-dependent kinases (CDK), their respective
cyclins, and inhibitors of CDKs (CKIs) were identified as instrumental components of the
cell cycle-regulating machinery. CDKs are enzymes that phosphorylate several cellular
proteins thus fueling the sequential transitions through the cell division cycle. In
mammalian cells the complexes of cyclins D1, D2, D3, A and E with CDKs are considered
motors that drive cells to enter and pass through the S phase. Cell cycle regulation is a
critical mechanism governing cell division and proliferation, and is finely regulated by the
interaction of cyclins with CDKs and CKIs, among other molecules (Morgan et al 1995).
It was also reported that CDKs have another key role the coordination of cell cycle
progression with responses to possible DNA-damage that could, if unchecked or unfixed,
lead to a lack of genomic integrity marking the onset of cell disease including cancers
(Huang et al 2006 in Science). The S-phase is thought to be the most vulnerable interval of
the cell cycle because during this interval all of 3 billion DNA bases of the human genome
must be replicated precisely in the sense of carbon copies being made of the existing DNA
strands, without any breaks in the sequence or base substitutions of the copied/replicated
strands. Therefore, this correct replication process controls the cells survival, especially
under genotoxic conditions such as those caused for example by mutagens or X-ray and
gamma-radiation. Furthermore, Huang et al. (2006) reported that CDK mediated the
phosphorylation of the FOXO1 transcriptional activator of the proapoptotic genes during
the S-phase; when DNA damage occurs either before or during the S-phase, a complex
network is activated in the cell which silences CDK thereby either delaying or
stopping/arresting the cell cycle progression. This may allow the cell to repair the DNA
damage by recombination involving BRCA2 and survive. However, if this is not possible
because the DNA damage was too great/irreparable, then FOXO1 would trigger apoptosis
(cell death). It was proposed that during the unperturbed (normal) S-phase CDK2
phosphorylates FOXO1 at the Serine
249
residue in the cell nucleus, which then results in the
transfer and sequestering of the FOXO1 in the cytoplasm, where it is well--separated from
the proapoptotic genes, the target of FOXO1 action.
Moreover, the CDK-mediated phosphorylation of BRCA2 during the unperturbed S-phase
renders inactive the DNA recombination. On the other hand, when DNA becomes damaged,
CDK2 is inhibited through the Cdc25A pathway, with the consequence of a dephosphorylated
FOXO1 which then remains in the cell nucleus and is able to activate the proapoptotic genes,
unless BRCA2 is able to induce DNA recombination and repair in time to prevent apoptosis.
The steps that follow are then as explained above: either DNA repair and continued cell
cycling, or apoptosis induced by FOXO1. There are still several important questions regarding
the entire process that need to be answered before the FOXO1 and CDK2 mechanisms of
action can be translated into successful clinical trials based on such knowledge.
A positive correlation has been noticed between over-expression of several cell--cycle
proteins and unfavorable prognoses and outcomes in several different cancer types (van
Diest et al 1995; Fukuse et al 2000). In human lung tumors and soft tissue sarcomas, it was
discovered that cyclin A/cdk2 complex expression and kinase activity were reliable
predictors of proliferation and unfavorable prognosis, thereby further substantiating the
epidemiological factors of cyclin signaling (Dobashi et al 2003; Noguchi et al 2000).


485

Fig. 1. Gene database of Cyclin-D1; Source: PBD website:
http://www.dsi.univ-paris5.fr/genatlas/fiche.php?symbol=CCND1
4.2 The p27 and p21 proteins
The proteins p27 and p21 were reported to be implicated in cyclin regulation and cancer
development (Fig. 2). Mouse embryonic fibroblasts that were deficient for p27 and p21 were
found to contain less cyclin D1 (Hashemolhosseini S, Nagamine Y, Morley SJ, et al., 1998).
and D2 (Cheng et al 1999) as well as cyclin D3 (Bagui et al 2000) than controls. Similarly,
mammary glands of p27-deficient mice were shown to possess decreased cyclin D1 levels
(Muraoka et al 2001). It has been demonstrated in vivo that p27 is necessary for maintaining
proper levels of cyclins D2 and D3, and this dependency on p27 is common to a wide
variety of cells/tissues in vivo. Regarding the molecular interaction between p27 and D-
cyclin, CDK4 is a clear candidate as a mediating molecule (Bryja et al 2004). Cells employ
CDK4/6 cyclin D complexes to flexibly titrate p27 from the complexes containing CDK2,
and thereby they control their proliferation. However, mutual dependency between cyclin D
and p27 serves also some yet unidentified function in differentiation-related processes.
Thus, loss of p27 not only causes unrestricted growth due to inefficient inhibition of CDK2
cyclin E/A, but may also elicit a decrease in levels of D-type cyclins, resulting in
differentiation defects. Upon ablation of cyclin D, cells lose their ability to titrate p27 from
CDK2cyclin A/E complexes and proliferation is suppressed. However, defects in
differentiation caused by the absence of D-cyclin are reminiscent to defects produced by the
absence of p27 (Bryja et al 2004). When the changes in levels of p27 and/or D-type cyclins
occur, an equilibrium alteration could result between proliferation/differentiation processes
that may in the end result in tumorigenesis (Bryja et al 2004).
4.3 D1 vs. E- cyclins
The D-type and E-type cyclins control the G1 S phase transition during normal cell
cycling and are important components of steroid- and growth factor-induced mitogenesis in
breast epithelial cells (Sutherland and Musgrove, 2004). Cyclin D1 null mice are resistant to
breast cancer that is induced by the neu and ras oncogenes, which suggests a pivotal role for


486
cyclin D1 in the development of some mammary carcinomas (Sutherland and Musgrove,
2004). Cyclin D1 and E1 are usually overexpressed in breast cancer, with some association
with adverse outcomes, which is likely due in part to their ability to confer resistance to
endocrine therapies. The consequences of cyclin E overexpression in breast cancer are
related to cyclin Es role in cell cycle progression, and that of cyclin D1 may also be a
consequence of a role in transcriptional regulation (Sutherland and Musgrove, 2004). One
critical pathway determining cell cycle transition rates of G1 S phase is the cyclin/cyclin-
dependent kinase (Cdk)/ p16Ink4A/ retinoblastoma protein (pRb) pathway (Sutherland
and Musgrove, 2004). Alterations of different components of this particular pathway are
very ubiquitous in human cancer (Malumbres and Barbacid, 2001). There appears to be a
certain degree of tissue specificity in the genetic abnormalities within the Rb pathway. A
model relating Rb to cyclin control in the overall scheme of pro-apoptotic behavior is shown
in Fig. 2.
In breast cancer these abnormalities include the over-expression of cyclins D1, D3 and E1,
the decreased expression of the p27Kip1 CKI and p16Ink4A gene silencing through
promoter methylation. These aberrations occur with high frequency in breast cancer, as each
abnormality occurs in ~40% of primary tumors. This fact implicates a major role for the loss
of function of the Rb pathway in breast cancer. Cyclin D1 is the product of the CCND1 gene
and was first connected to breast cancer after localization of the gene to chromosome 11q13,
a region commonly amplified in several human carcinomas, including ~15% of breast
cancers (Ormandy et al 2003). The fact that cyclin D1 was overexpressed at the mRNA and
protein levels in 50% of primary breast cancers have caused cyclin D1 to be considered one
of the most commonly over-expressed breast cancer oncogenes (Gillett et al 1994). Although
cyclin E1 locus amplification is rare in breast cancer, the protein product is overexpressed in
over 40% of breast carcinomas (Loden et al 2002). Cyclin D1 is pre-dominantly
overexpressed in ERC tumors, and cyclin E overexpression is confined to ER tumors (Gillett
et al 1994; Loden et al 2002). The overexpression of several cell cycle regulators has been
strongly associated with apoptotic-like behavior, as well as frank apoptosis, in cancer cells,
which include c-Myc, E2F-1 and HPV. Apoptosis and its connection to cell cycle-related
proteins is of interest therapeutically, as these types therapies could ultimately lead to the
cancer cell annihilation via apoptosis. Recently, a shift has occurred, changing the focus of
chemotherapy from exploration of agents that cause cell growth arrest to those that favor
apoptosis.
5. Biomedical applications of microarrays in clinical trials
5.1 Microarray applications to gene expression: identifying signaling pathways
Changes in homeostasis can be followed through various experimental strategies that
monitor gene expression profiling, for example, by employing high-throughput

microarray
technology. This section discusses briefly the successful use

of microarray technology in
RNA expression studies aimed at identifying signaling pathways that are regulated by key
genes implicated

in carcinogenesis/ tumorigenesis. A primary objective of tumor-profiling

experiments is to identify transcriptional changes

that may be the cause of the transition

from the normal to the tumor phenotype. Such changes may, however, occur also as a
consequence of various neoplastic transformation(s). More importantly, this approach may
allow

the identification of molecular fingerprints that can be utilized for

the classification of
different tumor types, and are therefore valuable diagnostic molecular tools in cancer


487

Fig. 2. Pro-Apoptotic Cancer Cycling Model: an update based on the previous model of
Aguda et al. (2003).
patients. For example, Alizadeh et al. (2000) have successfully used such an

approach to
identify molecularly distinct subclasses of diffuse

large B-cell lymphoma that could not be
distinguished by conventional diagnostic

tools. In another study, a molecular fingerprint

comprising approximately 50 genes has been isolated from a total of

over 6,000, and this
fingerprint can reliably differentiate between acute myeloid

leukemia and acute
lymphoblastic leukemia Golub et al (1999).


488
5.1.1 Identification of specific transcriptional targets in cancer
The approach requires, however, multiple independent experiments

with several large
groups of samples in order to enable one to reliably and reproducibly separate the
biologically relevant

changes from false ones that may occur as a result of the

genetic
heterogeneity between individual samples from the same tumor, for example. The two
examples quoted above were able to reproducibly identify tumor type-specific

molecular
determinants through multiple experiments with various tissue samples.
A different experimental approach to the one presented above is, however, needed for
identifying specific targets such as defined genes that are implicated in cancer progression;
this involves monitoring changes in transcriptional profile that occur as a result of
modulation of the expression level of the defined gene, or genes, selected for such studies.
The altered expression profile can be viewed as a blueprint by which the defined gene
controls its cellular function. The transcriptional profiles are thus employed to define

downstream signaling pathways that have been previously validated

through other techniques
such as

differential display Tanaka et al (2000) and serial analysis of gene expression

Yu et
al. (1999). This approach combined with microarray technology allows the simultaneous
identification of all potential targets. Its only drawback is the reliance upon the prior
knowledge of the selected genome for such investigations. The caveat is, however, that the
investigator who employs this approach needs also to devise additional experiments in
order

to confirm that genes identified with the microarray are indeed physiologically

relevant
targets.

5.1.2 Identification of downstream transcriptional targets of the BRCA1 tumor-
suppressor gene
The breast and ovarian cancer susceptibility gene BRCA1 is probably the most studied gene
in the breast cancer field because of its clinical significance and multiple functions. BRCA1
was shown to be mutated in the germ line of women

with a genetic predisposition to either
breast or ovarian cancer Mikki et al (1994). Most

mutations identified reported have resulted
in the premature truncation

of the BRCA1 protein. BRCA1 is known to encode a 1863 amino
acid phosphoprotein that is

predominantly localized to the nucleus, presumably with a
unique function. Protein sequence analysis

identified a C-terminal BRCT motif, which was
then postulated

to play a role in cell cycle checkpoint control in response

to DNA damage
Koonin EV, Altschul and Bork (1996). Consistent with this postulated role, BRCA1

becomes
hyperphosphorylated in response to various

agents that damage DNA such as /X--ray-
irradiation, an effect that was reported to be partially mediated by chk2 kinases

(Lee et al.
2000). Furthermore, BRCA1 has been shown to be implicated in at least three functional

pathways:
Mediating the cellular response to DNA damage,
Acting as a cell cycle checkpoint protein, and
Functioning in the regulation of

transcription.
However, the physiological significance of such BRCA1 actions as well as their relationships
with the function of BRCA1 as a tumor-suppressor

gene still remain to be defined. Further
details are presented next.
The BRCA1-BARD1 ubiquitin ligase
As shown above the BRCA1 gene encodes a 1863-amino-acid protein (Miki et al 1994) which
consists of a RING-finger domain in its terminal N-region, a region that includes a nuclear


489
localization signal and a domain that binds to many cellular proteins, and tandem BRCT
domains in its C-terminal region. BRCA1 is associated with a diverse range of biological
processes, such as DNA repair, cell cycle control, transcriptional regulation, apoptosis and
centrosome duplication. Thus, a specific role has already been postulated for BRCA1 in
transcriptional

regulation. The C-terminal domain of BRCA1 was reported to contain

a
potent transactivation domain when this was fused to a heterologous

DNA binding motif
(Monteiro, August and Hanafusa, 1996). The oligonucleotide array-based expression
profiling described above in Section 2.2 was employed by Haber (2000)

in collaboration with
Affymetrix Co. to identify the downstream transcriptional

targets of the BRCA1 tumor-
suppressor gene in order to define

its function (Harkin et al 1999). A known biochemical
function of BRCA1 is its E3 ubiquitin ligase activity. The following reported observations
provide only indirect, additional clues to the tumor-suppressor gene function of BRCA1.
Germ line mutations of BRCA1 were reported for half of breast-ovarian

cancer pedigrees
and for approximately 10% of women with early onset

of breast cancer, uncorrelated with
their family history (Fitzgerald et al 1996). It was also shown in other studies that somatic

inactivation of BRCA1 is rare in sporadic breast cancers (Futreal P, Liu Q, Shattuck-Eidens D
et al., 1994) and mutations were reported for approximately 10% of

sporadic ovarian
cancers, therefore suggesting potentially distinct genetic

mechanisms for sporadic, breast
and ovarian cancers (Berchuk et al 1998). The reduced BRCA1 protein expression reported
for the majority

of sporadic breast cancers indicates that epigenetic mechanisms

such as those
described in Section 6 was suggested to play a significant role in regulating the BRCA1
expression (Wilson et al 1999). Furthermore, a defect was reported in the transcription-
coupled

repair of oxidative-induced DNA damage in mouse embryo fibroblasts

with
attenuated BRCA1 function (Gowen et al 1998); this observation would suggest that BRCA1
plays a more general role in mediating the cellular response

to DNA damage. Thus, BRCA1
has also been reported to be involved in cell cycle checkpoint control, by

becoming
hyperphosphorylated during late G
1
and S cell phases, and then changing to transiently

dephosphorylated early after the M phase (Ruffner and Verma, 1997). Moreover, the BRCA1
overexpression has been reported to induce a G
1
/S arrest in human colon

cancer cells
(Somasundaram et al, 1997). By comparison with the cancer regulation model in Figure 2, it
seems very significant for oncogenesis that BRCA1 is physically associated with the
transcriptional

regulators p53 (Ouichi et al 1998), CtIP (Yu et al 1998), c-Myc (Wang et al
1998), as well as the histone

deacetylases HDAC1 and HDAC2 (Yarden and Brody 1999).
The physical association of BRCA1 with c-Myc acquires special significance as c-Myc seems
to be involved in controlling telomerase activity, whereas p53 is involved in DNA-repair,
cell-cycling and apoptosis. Therefore, in the simplified model presented in Figure 3, one
should add the BRCA1 links to both p53 and c-Myc in order to facilitate an understanding of
the BRCA1 possible roles in oncogenesis.
5.1.3 Selecting gene expression systems
There are several related problems in studying gene function by expression profiling. For
example, it has been often reported to be difficult to generate cell lines that overexpress
genes such as BRCA1, or p53, because their forced overexpression can lead either to
growth suppression or apoptosis (as shown for example in Figure 3, and at the end of the
previous section). However, in the case of BRCA1, it was reported that the tet-off inducible
expression system

(Gossen and Bujard 1992) can be utilized to generate cell lines with
highly regulated inducible

expression of BRCA1 (Harkin et al, 1999). This inducible


490
expression system introduces into the cells a chimeric transactivator; the latter

consists in
the tet repressor fused to the VP16 transactivation

domain. This chimeric transactivator is
inactive in the presence

of tetracycline, whereas in the absence of tetracycline it can bind
to

promoters that contain the tet operator sequence; the latter sequence is then

utilized to
drive the expression of BRCA1. This expression system has a major advantage in that it
allows the

change in just one parameter involved in the induction of BRCA1. The BRCA1
induction in one population is the only difference between the genetic backgrounds of the
two populations that are being compared by oligonucleotides arrays. A number of BRCA1
transcriptional

targets can thus be identified with Affymetrix oligonucleotides arrays, and
among these, the

stress and DNA damage-inducible gene GADD45 was the gene that
exhibited the greatest degree of differential signal intensity (Harkin et al, 1999). The
specific target genes thus identified were also verified by Northern blot or quantitative
reverse transcriptase-PCR analysis in order to confirm induction

in response to the
stimulus, that is, the induction

of BRCA1 (Harkin et al, 1999). Total RNA was extracted
from cells in which the exogenous BRCA1 was either switched off (+ tet) or switched on (
tet). Fluorescent images were generated using the Affymetrix human cancer G110 array
containing approximately 1,700 genes that were previously reported to be implicated in
cancer; such fluorescent images were then scanned and analyzed. Two lanes were present
in such images that corresponded to individual arrays hybridized with biotinylated cRNA
probes and were generated from cells in which exogenous BRCA1 was either induced (+
tet) or repressed (- tet). Each gene on the array was represented by 16 probe pairs, one
being wild-type and one containing a mismatch at the central nucleotide. In such
fluorescent images, two genes, GADD45 and ATF3 were identified (and confirmed by
Northern blot analysis) as being the transcriptional targets of the BRCA1 tumor-suppressor
gene. Furthermore, in this BRCA1 study, the induction of GADD45

by BRCA1 was
reported to be correlated with the BRCA1-mediated activation of the

c-jun N-terminal
kinase/stress-activated protein kinase JNK/SAPK

pathway. Significantly, the activation
of JNK/SAPK was then shown to be

required for the BRCA1-mediated apoptotic cell
death in this cell

line system. This finding suggests an interesting model for the BRCA1-
mediated

apoptosis, as presented in some detail by Harkin et al (1999). Most significantly,
the experimental approach reported by Harkin et al (1999) was indeed able to define
physiologically relevant target genes. In another recent report, Yu et al (2001) utilized a
modified version

of the tet-off inducible expression system to define the downstream

transcriptional targets of the p53 tumor--suppressor gene (Yu et al 1999).

A total of 34
genes were identified that exhibited at least

a 10-fold upregulation in response to the
inducible expression of

p53. Somewhat surprisingly, there was a marked heterogeneity

of
the response when it was evaluated in different cell lines derived

from the same tissue of
origin. Among the 33 genes studied

only nine were found to be induced in a panel of five
unrelated

colorectal cell lines, and 17 were induced in a subset; eight

were not induced at
all in any of the five cell lines examined. This

can be interpreted as being due to a high
degree of cell type specificity. Furthermore, p53 was not absolutely

required for induction
-- for the majority of the genes identified-- in response to either adriamycin or 5-FU.
Therefore, these agents do not seem to act exclusively through p53, suggesting that there
is inherent redundancy in the majority

of signaling pathways. Such inherent redundancy
in signaling pathways of cancer, and untransformed, cells might be important in
understanding the results of clinical trials in cancer treatment with signal transduction
modulators that will be discussed in the next subsection (5.2).


491
5.2 Clinical trials with signal transduction inhibitors -- novel anticancer drugs active
in chemo-resistant tumors
Recently, there is an increasing number of reports suggesting that human cancers frequently
involve pathogenic mechanisms which give rise to numerous alterations in signal
transduction pathways. Therefore, novel therapeutic agents that target specific signal
transduction molecules or signaling pathways altered in cancer are currently undergoing
clinical trials often with remarkable results in cancer treatments of patients in which chemo-
and/or radiotherapy resistant tumors have become apparent. For example, several new
classes of such anti-cancer drugs are:
tyrosine/threonine kinase inhibitors, including: STI-571
(Gleevec, or Imatinib Mesylate), ZD-1839 (Iressa), OSI-774, and flavopiridol, which
are ATP-site antagonists and have recently completed phase I and phase II trials (see for
example, Liu et al, 1999);
several other kinase antagonists that are currently undergoing clinical evaluations,
including UCN-01 and PD184352;
other strategies for downmodulating kinase-driven signaling include 17-allyl- amino-17
demethoxygeldanamycin and rapamycin derivatives. Phospholipase-directed signaling
may also be modulated by alkylphospholipids.
Farnesyltransferase inhibitors, originally developed as inhibitors of ras-driven signals,
may attain activity by affecting other/or additional targets (see for example, Zujewski,
Horak, Bol, et al., 2000; End, Smets, Todd, et al., 2001).
monoclonal antibodies Herceptin and C225.
Signal transduction is an efficient method for fine-tuning the development and modeling of
cancer treatments (Ideker at al., 2001, 2002). There is also a detailed NCI report on clinical
trial and signal transduction modulators as novel anticancer (Sausville, Elsayed, Monga and
Kim, 2003).
5.3 Interactome-transcriptome analysis and differential gene expression in cancer
It has been claimed that high-throughput yeast-two-hybrid (HT-Y2H) methods will allow a
systematic approach to functional genomics, by placing individual genes in the global
context of cellular functions (Mendelsohn and Brent, 1999). One finds that high-throughput
screening methods such as HT-Y2H have indeed allowed the mapping of the first
interactomes for three eukaryotes (Giot et al 2003; Li et al 2004; Uetz et al 2000). Because of
the human interactomes much larger size and its very high-degree of complexity there will
be quite high costs and labor involved in obtaining the data necessary, for example, for an
HT-Y2H mapping of a complete human cell interactome. Furthermore, the complete data
analysis together with the assembly of the complete interactome network is likely to require
both conceptual and computational advances, in addition to a significant amount of time
and collective effort(s) by one or several research teams. In view of the high, potential
importance of the human interactome for cancer therapy, and also for improved diagnosis
and rational clinical trials, such an effort should now be a top priority. Such an effort
should also be coordinated with an improved mapping of the complete yeast interactome as
a model, or test, system. Meantime, there have been since 2005 a few reports of surrogate,
or partial, human cancer cell interactomes in the form of predicted maps of human protein
interaction networks based on partial data and comparative analysis. Such studies
emphasize even further the need and urgency for the complete mapping of several human


492
cancer cell interactomes. Following the seminal studies of DeRisi et al (1996) that utilized
cDNA microarray to analize gene expression patterns in human cancer, there have been
relatively few attempts at deriving hypothetical gene expression patterns in human cancer.
The first claim of such an attempt was recently made by Wachi, Yoneda and Wu (2005) for
genes that were differentially expressed in squamous cell lung cancer tissues from five
patients who had undergone surgical removal of the tumor(s). cRNA samples were
prepared and hybridized to arrays obtained from Affymetrix (Hg-U133A
TM
.). These
authors were able to carry out paired t-test analyses for each individual patient in order to
distinguish the genes in which expression levels in their squamous lung cancer cells differed
from the paired normal lung tissue (control samples) obtained from the same five
individuals. The authors prediction methodology will be briefly discussed in the next
subsection as some of the details are relevant for the evaluation of these results which were
the first to be reported for the (hypothetical) interactometranscriptome analysis of human
cancer cell data for a group of five patients with the same diagnosed form of (lung) cancer,
and with the same treatment (tumor removal by surgery). The hypothetical human protein
interaction maps are a relatively new endeavor (Brown and Jurisica, 2005; Lehner and
Fraser, 2004) perhaps because they are likely to have many false positives, as well as miss a
significant fraction of the relevant/real protein-protein interactions. Currently, microarray
analysis still suffers inherently from relatively high noise levels and the accompanying
information loss (buried in noise); although this inherent noise problem is partially
eliminated through multiple replicate analyses, the number of replicates is often limited by
the availability and the material cost. Another significant problem of such microarray
projects is the huge amount of data that needs to be processed in order to obtain useable
information (Claverie, 1999).
5.3.1 Analysis of human protein-protein interactions (HPPI) and integration of array
data into a predicted protein-protein interaction network (PPIN)
Wachi, Yoneda and Wu (2005; WYU05) employed for their human cell data analysis a web-
presented database (OPHID, April 25, 2005) of predicted interactions between human

proteins (Brown and Jurisica, 2005) based on data for human and other four organisms
which included the intensely-studied yeast and fruit fly. (OPHID is freely available to
academic users at http://ophid.utoronto.ca). This protein interaction database listed 16,034
known human protein interactions obtained from various public protein interaction
databases, as well as 23,889 additional, predicted interactions which are evaluated using
protein domains, gene co-expression

and Gene Ontology terms. The results can be
visualized in OPHID using a customized,

graph visualization program. The data comprises
literature-derived human PPI from

BIND, HPRD and MINT, with predictions made from
Saccharomyces

cerevisiae, Caenorhabditis elegans, Drosophila melanogaster

and Mus musculus.
The genes in the WYU05 array were matched to those in OPHID using gene symbols and
protein sequences. In this manner, 2137 genes in the WYU05 microarray experiments were
matched to the protein network from OPHID. These predictions should, however, be
thought

of only as hypotheses until they are experimentally validated. On the other hand,
there is increasing

evidence that at least certain PPIs may be conserved through evolution
(Pagel et al 2004; Wuchty et al 2003). Recently, Sharan et al (2005) claimed that about 50% of
the protein-protein interactions predicted by using interologs between microorganisms are
also experimentally validated. The interologs approach might play therefore a role in the


493
partial validation of the HT-Y2H protein network mapping without, however, necessarily
achieving the claimed, global validation of the predicted (hypothetical) interactome.
Differentially expressed genes (DEGs) from squamous cell carcinomas (SCCs) were then
identified as discussed above and their connectivity in the network graph was examined to
determine their topological properties, such as the edge distribution for DEGs in
comparison with the surrounding graph subnetwork.
5.3.2 Differentially expressed genes DEG- results for SCC of human lung
The genes that are upregulated in SCC were found to exhibit a positive correlation
(Pearsons r-coefficient of 0.82) with the number of edges associated with them (Fig. 1a of
Wachi, Yoneda and Wu 2005), which was interpreted as indicating that DEGs that are
upregulated in SCC are also highly connected. However, the downregulated genes were
reported also to have a positive correlation (r = 0.75) to connectivity, albeit slightly lower
(Fig. 1b of Wachi, Yoneda and Wu, 2005). On the other hand, microarray probesets that
matched the genes in the protein network (n =-2,137) had a negligible correlation coefficient
(r =0.06) to link number, proving that the genes on the test microarrays did not contribute to
bias in the number of links for DEGs in SCC.
A k-core analysis of DEGs in SCC of the human lung was also carried out (loc. cit.) which
were reported to measure how close are the DEGs to the topological center of the human PPI
network. Based on the k-core analysis, it was concluded that: the upregulated genes are more
centrally located in the protein network than the down-regulated genes. If duplicated and
validated, such studies would be important as the topological centrality of the genes in the
interactome was previously reported to be associated with the essential functions of the
genes in the yeast (Jeong et al 2001). Such essential genes, are lethal when mutated, and also
tend to have high connectivity. Moreover, other genes that are not essential in this sense, but
provide a vital function in toxin metabolism were reported to have a high number of edges
associated with the nodes, and to be less well connected than the essential genes in yeast
(Said et al 2004). Furthermore, a k-core analysis has also been performed on the yeast
essential genes and they were reported to be global hubs, whereas the non-essential genes
were not hubs (Wuchty and Almaas, 2005). It was also claimed that these essential, global
hubs are conserved throughout different species; however, one notes that, thus far, there is
insufficient data and evidence to prove this claim, or hypothesis. Nevertheless, one may
consider as a working hypothesis that there should be a core set of genes that needs to be
maintained throughout the course of somatic evolution in the tumor microenvironment (Wachi,
Yoneda and Wu, 2005). This hypothesis is thus consistent with the somatic evolution model of
cancer. Such conserved genes might be the essential genes in cancer cells, and they may
also have somewhat analogous to the global hub, essential genes reported in yeast (Wuchty
and Almaas, 2005). DEGs would thus be essential for the survival and proliferation of cancer
cells in SSC of the human lung, and the upregulated genes would be centrally located in the
protein network as well as have higher connectivity, perhaps suggesting their possible
essential role(s) in human (SSC) lung cancer. As this is the first report of a predicted/
hypothetical human cancer interactome network one should definitely consider replicating
the reported studies and also evaluating such potentially important findings in the context
of a complete human cancer interactome (differential) analysis. This possibility that DEGs
might be essential for the survival and proliferation of cancer cells in SSC of the human lung
has much too important consequences to be ignored; therefore, it must be thoroughly


494
investigated and also tested with sufficiently extensive, translational genomics and
transcriptional databases that do not seem to be currently available (Han et al. 2006). Further
supporting analyses for this conjecture made by Wachi, Yoneda and Wu (2005) are
considered in the next section.
5.4 Cancer proteins and the global topology of the human interactome network
A recent and extensive study of both cancer and non-cancer proteins (Jonsson and Bates,
2006) was integrated into a validated protein-protein interaction (PPI) network, or
interactome, of human proteins. In their report, the connectivity properties were
investigated for all proteins previously shown to be modified as a result of mutations
leading to cancer (Furteal, et al 2004). A global protein-protein interaction network was then
constructed by a homology--based method which is claimed to accurately predict protein-
protein interactions. It was then suggested that human proteins that are involved in cancer,
or cancer proteins, exhibit a network topology which is substantially different from that of
other proteins which are considered not to be involved in cancer. Notably, increased
connectivity was pointed out for cancer proteins involved in the following subnetworks: cell
growth and apotosis-related, signal transduction (MAPK, TGF-beta, insulin, T-cell and B-cell
receptor, adipocytokine, cytokine-cytokine interaction), cell motility/cytoskeleton, cell
communication, adherence junction, focal adhesion, leukocyte migration, antigen processing
and folding/sorting/degradation. Furthermore, it was proposed that such observations
indicate an underlying evolutionary pressure to which cancer genes, as genes of central importance,
are subjected. Linking these claims with previous proposals by Wuchty and Almaas (2005)
that globally central proteins form an evolutionary backbone of the proteome and are essential
to the organism, (and also with the conjecture made by Wachi, Yoneda and Wu, 2005,
discussed here in Section 5.3.), Jonsson and Bates (2006) suggested that cancer proteins may
generally be older than the non-cancer ones in evolutionary age. Furthermore, they also
suggested that the somatically mutated cancer proteins may be of somewhat younger
evolutionary average age in comparison with those from the germline, as a consequence of
the evolutionary selection pressure postulated to affect germline mutated proteins. Note
also that the previous study of (SCC) human lung cancer by Wachi, Yoneda and Wu (2005)
also reported increased interaction connectivity in differentially expressed proteins in
human lung cancer tissues.
6. Epigenomics in mammalian cells and multi-cellular organisms
6.1 Epigenetic controls
Upon completion of the US Human Genome Mapping Project and related studies, it became
increasingly evident that a sequence of 30,000 or so active genes that encode and direct the
biosynthesis of specific proteins could not possibly exhaust the control mechanisms present
in either normal or abnormal cells (such as, for example, cancer cells). This is even more
obvious in the case of developing embryos or regenerating organs. Subsequently, more than
120,000 genes were suggested to be active in the human genome (Nature, 2004).
Furthermore, specific control mechanisms of cellular phenotypes and processes were
recently proposed that involve epigenetic controls, such as the specific acetylation <=> de-
acetylation reactions of DNA-bound histones (for an overview article on epigenomics see,
for example, Scientific American 2003, December issue). Such controls intervene from outside
the genome but ultimately they also affect gene expression. Therefore, gene profiling


495
techniques would need to be combined with epigenomic tools and analysis in order to gain
an improved understanding of functional genomics and interactomics. Epigenomic tools
and novel techniques begin to address the complex and varied needs of epigenetic studies,
as well as their applications to controlling cell division and growth. Such tools are, therefore,
potentially very important in medical areas such as cancer research and therapy, as well as
for improving domestic animal phenotypes without involving genomic modifications of the
organism. This raises the interesting question if epigenomically controlled-growth
organisms (ECGOs) -- to be produced in the future-- would be still argued against by the
same group of people who currently objects to GMOs, even though genetic modifications
would be neither present nor traceable in such ECGO organisms?
6.2 Novel tools in epigenomics: rapid and ultra-sensitive analyses of nucleic acid
protein interactions
Several novel techniques could also be applied for the highly-selective detection of
epigenomic changes in mammalian cells related to diseases such as individual types of
cancer (Jones and Laird, 1999; Plass, 2002) and Alzheimer disease. Such novel tools are
likely to be utilized in a wide range of applications in biotechnology research related to
Post-Genomics and Epigenomics. Tumor suppressor genes are transcriptionally silenced
by promoter hypermethylation that also appears to lead to alterations in chromatin
structure- a possible mechanism for such repression of the suppressor genes. In contrast
to the genetic mutation or deletion mechanism of tumor suppressor gene inactivation,
epigenetic inactivation of tumor suppressor genes would occur via methylation of specific
DNA regions that could be prevented by DNA methyl-transferase or histone deacetylase
inhibitors. Aberrant CpG--island methylation has non-random/tumor-type-specific
patterns (Costello et al 2000). Such patterns can be identified by employing methylation--
specific PCR (MS-PCR; Herman et al 1996), and can also be employed either for tumor
class prediction by microarray-based DNA methylation analysis (Adorjan et al 2002) or
for high-throughput microarray-based detection and analysis of methylated CpG islands
(Yan et al 2002). Hypermethylation profiling is important for both accurate diagnosis and
the development of optimal strategies in cancer therapy. Gene promoter
hypermethylation has been reported in both tumors and serum of patients diagnosed with
several types of cancer: head and neck cancers (Sanchez-Caspedes et al. 2000),
nasopharyngeal carcinoma (Wong et al. 2002), non-small cell lung cancer (Belinsky et al
1998; An et al 2002), gastric carcinoma (Lee et al 2002), liver, prostate, bladder and
colorectal cancers (Wong et al 1999; Jeronimo et al 2002). Substantial efforts are being
made recently for the development of new methods and tools that are capable of sensitive
and quantitative DNA methylation analysis, as well as early and accurate diagnosis of
cancer. Among such tools are: Fluorescent methylation--specific polymerase chain
reaction assay (FMS-PCR; Goessl et al 2000), SNIRF (Mahmood and Weissleder, 2003),
indocyanine green-labeling (IGL) for human breast carcinomas (Ntziachristos et al 2000),
ConLight-MSP (Rand et al 2002), COBRA (Xlong and Laird, 2002), Methylation-Sensitive
Single Nucleotide Primer Extension (Ms-SnuPE; Gonzalgo and Jones, 1997), DNA
microarray sensitive detection by Metal-Enhanced Fluorescence (MASD/MEF; Lakowicz,
2001; Malicka et al 2003 a, b)), and NIR Fluorescence Micro-Spectroscopy (NIRFMS),
single cancer cell detection (Baianu et al 2004a). Specific molecular markers of cancer
(Sidransky, 2002) hold the promise to identify those molecular signatures that are unique
to specific types of cancer, and are essential for the early accurate diagnosis and treatment of


496
cancer. Such novel molecular tools and methodologies could be employed to rapidly and
accurately identify molecular signatures of cancer and aging-related diseases in
mammalian cells in culture in order to determine how specific epigenomic mechanisms
involved in the control of cell division and apoptosis operate throughout the cell cycle.
Among the specific epigenomic control mechanisms that one could investigate with such
new tools are: CpG-island methylation, p15 (INK4b) and p16 (INK4a) hyper-methylation
(in synchronous hepatic carcinoma cells), GSTP1 methylation in non-neoplastic/
synchronous cells, as well as histone-deacetylation and its effects on histone- nucleic acid
interactions in stable synchronous cell populations in culture. Both cancer and aging were
reported to involve DNA methylation of specific genome regions (van Helden & van
Helden, 1989; Ahuja et al 1998). Gene expression profiling and epigenomic testing could
be carried out with both ultra-sensitive, novel human and mouse microarrays. Powerful
spectroscopic and microspectrosopic techniques can be then employed for the analysis
and further improvement of such tools for the investigation of nucleic acid--protein
interactions.

High-field 2D NMR of protein--protein and protein--nucleic acid interactions
NIR Chemical Imaging of protein clusters in cells and single cancer cells in tissue;
NIR-FMS; SNIRF
MEF and FCS/FCCS/ FRET detection of single molecules amplified-ELISA; NASBA
Ms-SnuPE; FMS-PCR; Lux
TM
Fluorogenic Primers*/ RT-PCR*,
MyArray
TM
DNA- Human*, GeneFilters
R
Human Regular Arrays**.
Specific Knock-out or silencing shRNAis (SuperArrayTM).
**The testing of these new tools can be carried out for example with stable and synchronous
mammalian (human HeLa and mouse) cells in culture.
Table 1. Techniques under Development and Related Applications that are commercially
supported *,**.
7. Conclusions and discussion
Novel translational oncogenomics research is rapidly expanding with a view to the
application of new technologies, findings and computational models in both pharmaceutical
and clinical areas. Sample analyses in recent clinical studies have shown that gene
expression data can be employed to distinguish between tumor types as well as to predict
outcomes. Important, potential applications of such results are individualized human cancer
therapy (Pharmacogenomics) and personalized medicine. There is clearly a need for
individualized cancer therapy strategies based on high-throughput microarray information
recorded for isolated tumor cell lines from stage I through stage III cancer patients. Studies
of Differential Gene Expression in human cancer cell lines are clearly required for
developing new strategies for efficient cancer therapies for patients whose tumors have
developed resistance to existing therapies. Such gene profiling expression, proteomic,
interactomic and tissue array data is essential for improving the survival rate of stage III
cancer patients undergoing clinical trials with novel signaling pathway inhibitors/ blocker
medicines, such as those discussed in some detail in Section 5. Several technologies aimed


497
at future applications in oncogenesis are currently under development both in the direction
of improved detection sensitivity and increased time resolution of cellular events, with the
limits of single molecule detection and picosecond time resolution already being reached
(Sections 2.5, 2.6 and 6.2). The urgency for funding and carrying out the complete mapping
of a human cancer interactome with the help of such novel, high-efficiency / low-cost and
ultra-sensitive techniques is pointed out for the first time in the context of recent findings by
translational oncogenomics and human cancer interactome predictions.
8. Acknowledgments
The author gratefully acknowledges receiving helpful suggestions, pertinent documentation
and critical comments from: Dr. Mark Band, Director of the Genotyping/ Transcriptomics
Unit at the Keck Center, Dr. Lei Liu, Director of BioInformatics Unit at the Keck Center,
Professor Schuyler Korban, and Professor James F. Glazebrook of the Mathematics Dept. at
UIUC and Eastern Illinois University, respectively. This research was partially supported by
Renessen Co., the IMBA Consortium, an USDA Hatch Grant No. ILLU-0995362 and AES at
UIUC.
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Part 7
Transcriptional Analysis

23
In-silico Approaches for RNAi
Post-Transcriptional Gene Regulation:
Optimizing siRNA Design and Selection
Mahmoud ElHefnawi
1
and Mohamed Mysara
2

1
National Research Center

2
The University of Nottingham
1
Egypt
2
UK
1. Introduction
RNA interference (RNAi) is a naturally occurring endogenous biological post-
transcriptional cellular mechanism that regulates against foreign genetic elements such as
viruses and inserted gene transcripts as well as in-house gene expression regulation. Small
interfering RNA (siRNA) molecules utilize this mechanism to promote homology
dependent messenger RNA (mRNA) degradation.
The utilization of siRNA as a molecular target to silence gene expression has been used
extensively as a research tool in functional genomics. The unprecedented advantage of
siRNA molecules, which is mainly related to the ability of effective and specific inhibition of
disease causing genes, elicited great expectations in therapeutic applications and drug
discovery. siRNAs potential as a drugs was investigated in viral and cancer models, and
showed successful results with diseases such as HIV, HCV and several types of cancer; as
most of these diseases have no cure. One advantage of siRNA-based drugs is their feasibility
in clinical trials following approval of phase 1. Moreover, they do not rely on an intact
immune system which give the advantage over other long double stranded RNA (dsRNA).
However, several factors challenge the design of selective siRNA molecules with highly
guaranteed silencing efficiency. Therefore, careful selection of siRNAs complying with all
necessary properties is crucial for efficient functional performance.
This Chapter discusses RNA interference using small interfering RNS (siRNA) starting with
the biological nature of mRNA and siRNA. Then it tackles factors contributing to siRNA-
mRNA silencing from both biological and bioinformatics aspects that should affect siRNA
effectiveness. Then, it represents step wise workflow for rational siRNA design considering
state of the art tools and algorithms. By the end of this chapter, various tools are presented
for siRNA evaluation phases that are used to predict siRNA efficiency and efficacy, with a
practical example applying the proposed methodology.
2. Small interfering RNA
Small interfering RNAs siRNAs are one of the cell defence mechanisms that act against not
only exogenous genetic materials like virus genes but also against cell endogenous genes as


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one of the post-transcription regulation method (Ullu et al. 2002). These natural-occurring
siRNAs target mRNAs (whether they are over expressed or abnormal) in a manner, so
selective and potent, that they became the core of interest of many biologists in the last
decade. Although siRNAs are not the only layer responsible for post-transcriptional
regulation, they have the advantage of hardly invoking the innate immune response
(Interferon-response) in contrast to long double stranded RNA (Stark et al. 1998) . In
addition, siRNAs, have shown to be very promising new therapeutic agents in various
diseases especially in Cancer, Aids and Neurodegenerative disorders as most of these
diseases have no cure (Hutvgner & Zamore 2002; Surabhi & Gaynor 2002; Xia et al. 2004).
That is why siRNA has been used as a drug for cancer clinical trials on human producing
the efficient and specific effect on human as it was expected (Davis et al. 2010).
2.1 siRNA mechanism of action
The mechanism pathway of siRNA is as follows: long dsRNA is cleaved by DICER a
ribonuclease III-type enzyme into the short molecules of siRNA duplexes, being
homologous to the mRNA targeted for silencing, siRNA triggers the formation of RNA-
induced silencing complex (RISC) in which the double stranded siRNA is incorporated
cutting the long double-stranded RNA molecules to double stranded small interfering RNA
(ds-siRNA), as illustrated in the [Fig. 1]. Then it is anwounded leading to single stranded
siRNA that binds to the target mRNA sequence resulting in its cleavage, and according to
the type of the RISC complex the RNAi action is directed through mRNA degradation,
action arrest or chromatin modification. [5]. This is detailed below:

Fig. 1. Small interfering RNA formed of two short stranded RNA sequences complementary
to each other.
Due to the homology (similarity) between the double stranded siRNA (ds-siRNA) and the
targeted messenger RNA (mRNA), the aggregation of a complex called RNA induced
silencing complex (RISC) is triggered. After binding with ds-siRNA, RISC acts to separate
(unwind) the strand making the sense and the antisense strands (passenger and guide
strand). After siRNA unwinding into small single strand, it could produce its action with
three different mechanisms [Fig 2].
2.1.1 Direct cleavage method
The single stranded RNA together with RISC bind to the targeted mRNA and induce its
degradation by the Ago-2 degradation (protein triggered by RISC-siRNA complex acts to
break the targeted mRNA). The degraded mRNA is finally digested with, what is called,
cellular lysosomes. This is the main mechanism by which siRNA causes selective and potent
gene silencing, but this only occurs in case of high level of similarity between siRNA and the
targeted mRNA region (Birmingham et al. 2006).
In-silico Approaches for RNAiPost-Transcriptional
Gene Regulation: Optimizing siRNA Design and Selection

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2.1.2 Seed-mediated translational attenuation
The complementation between the siRNA seeding region hexamer (from the second to the
seventh position) and the 3UTR (untranslated region) of the mature mRNA has been
identified capable of inhibition of that mRNA's translation and causing its degradation (E.
M. Anderson et al. 2008; Birmingham et al. 2006).

Fig. 2. Naturally occurring siRNA synthesis pathway and three its possible mechanisms of
action. Endogenous (naturally occurring) siRNA are produced from either microRNA or
long double strand RNA after their cleavage by the Dicer enzyme so they produce double
strand siRNA. Both endogenous and exogenous (introduced by researchers) ds-siRNA pass
through the activation process starting with unwinding and RNA induced silencing
complex (RISC) to the lead single strand siRNA. Then RISC- single strand siRNA complex
silence the targeted gene either by one of the three mechanisms: 1) Binding to the mRNA
leading to their breakage through Age2 mechanism. 2) Binding to the 3 end and mediate
translational attenuation of the mRNA. 3) Gene silencing through chromatin modification
[Figure from the work of (Dorsett & Thomas Tuschl 2004)].
2.1.3 Chromatin modification
siRNA has another mechanism of interference by chromatin modification as illustrated by
Dorsett and Tuschl in their description of Scherer work that siRNA is one of the three major
nucleic-acid-based gene silencing mechanisms (Dorsett & Thomas Tuschl 2004).


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3. Factors that affect siRNA design
In order to understand the interaction between siRNA and the targeted mRNA, several
factors have been known to affect the design of effective and specific siRNA. These factors
can be further sub classified into four major classes design as illustrated by Birmingham
(Birmingham et al. 2007). Firstly, Targeted region or what is called sequence space, this
section handles the identification of regions in the mRNA to be targeted by the designed
siRNA. This step is highly critical as targeting the wrong region would abolish the effect of
all designed siRNAs. Sequence space is affected by several factors: Transcript region,
Transcript size, mRNA multiple splicing, Orthologs consensus and Single nucleotide
polymorphism. Secondly, siRNA sequence space preparation, here we discuss internal
repeats, positional preferences, and other desirable/undesirable words/motifs are
discussed. Thirdly, siRNA thermodynamic properties and both siRNA and mRNA target
accessibility. It includes parameters like GC content, palindromes, in addition to
thermodynamic stability and differential ends stability which have been identified to be
highly important factors in siRNA selection. Forthly, siRNA specificity describing
mechanisms through which siRNA could invoke immune reaction or has off-target effect.
Each of these factors can greatly affect siRNA selection and therefore they should be studied
thoroughly.
3.1 Target sequence space [Targeted region preprocessing]
Targeted regions (or what is called sequence space) are areas of the mRNA that should be
assigned for targeting by the designed siRNA. There are five factors affecting the selection
of the proper sequence space summarized in (Birmingham et al. 2007).
3.1.1 Transcript regions and size
siRNA should target regions in the mRNA that is not affected by the maturation process,
hence targeting 3UTR, 5UTR and (most importantly) open reading frame (ORF) [Fig 3].
Normally both 3UTR and 5UTR could be excluded from targeted sequence space, unless
sequence space needs to be widened. If the mRNA length is < 500 nucleotides, 3UTR and
5UTR should be included in target space selection.

Fig. 3. Maturation process of premature to mature mRNA. This figure illustrates different
regions that vary due to omission and insertion during the maturation process. In the
maturation process omission of the introns (non coding areas) and addition of 5 cap and 3
tail) takes place (Mysara 2010).

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3.1.2 Multiple splicing and orthologs consensus
One mRNA could be coding for several proteins as the process of splicing is accompanied
by rearrangement of exons. There are several mechanisms of alternative (differential)
splicing as exon insertion or deletion but the main mechanism; as described in the work of
Black; is exon skipping (Black 2003). This phenomena form a huge obstacle if there is a need
to target all the mRNA transcripts; therefore, regions in common among them should be
recognized and targeted [Fig 4]. All the mRNAs transcripts should be included in target
space selection. In case of handling multiple organisms (as in global vaccines or rapidly
mutated species as virus) the consensus between different targeted mRNAs should be
considered in the target space selection.
3.1.3 Single Nucleotide Polymorphism (SNPs)
Single Nucleotide polymorphism (SNP) is very crucial in siRNA design where single
(several) Nucleotide(s) difference could cause dramatic shift in the produced protein (or in
its regulation) or could have a non-sensible effect in this case it is named silence
polymorphism. There are two main locations for SNPs existence non-coding and coding
regions [Fig 5]. The first region is the non-coding region, where SNP existing in the Introns
will not affect the mature mRNA, thus the siRNA targeting it. However, if the SNP is
located in the 3 UTR or 5 UTR, caution should be taken in cases where the siRNA is
designed to target them. The second region is coding region, where SNP exists in the
protein coding region (ORF or Exons), there are two possibilities: SNPs will not affect the
produced protein due to degeneracy of the genetic code, or it could cause changes in the
produced protein, hence siRNA targeting this region should be excluded.

Fig. 4. mRNA alternative splicing phenomena results in several transcripts from the same
gene. Each of these transcripts is later translated in a different protein. These proteins
functions could be similar or non-similar to each other.


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3.2 SiRNA sequence space [Positional/word preferences]
Positional/word preferences in the sense/antisense strand of the siRNA are a crucial
determinant of siRNA functionality. Several position dependant preferences were identified
from analysis of siRNA experimental dataset, which can affect siRNA selection process.
Among those preferences within the sense strand (Ui-Tei et al. 2004): (i) A/U at the 5 end of
the antisense strand;(ii) G/C at the 5end of the sense strand; (iii) at least five A/U residues
in the 5 terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of
more than 9 nt in length. (Reynolds et al. 2004): (I) At least 3 A/U bases at positions 1519
(sense strand). (II) Absence of internal repeats. (III) An A base at position 19 (sense strand).
(IV) An A base at position 3 (sense strand). (V) A U base at position 10 (sense strand). (VI)
A base other than G or C at 19 (sense strand). (VII) A base other than G at position 13
(sense strand). (Mohammed Amarzguioui & Prydz 2004): asymmetry in the stability of the
duplex ends (measured as the A/U dierential of the three terminal basepairs at either end
of the duplex) and the motifs S1, A6, and W19. The presence of the motifs U1 or G19 was
associated with lack of functionality.

Fig. 5. Classification of SNPs according to region of occurrence in the mRNA.
Several positions in siRNA duplex that could affect their efficiency, as in (Birmingham et al.
2007), candidate duplexes with five or more of any single base in a row, should be removed.
Although less detrimental than G/C stretches, repeated bases have also been shown to
reduce functionality. Stretches of repeated base-containing sequences are less selective, and
A or U/T stretches may additionally target regulatory motifs. Moreover, candidate duplexes
with more than six consecutive Gs and/or Cs stretches of Gs and Cs have been shown to
be one of the strongest negative determinants for siRNA activity that should be removed.
Such regions have pronounced local stability, greatly inhibiting duplex dissociation. In
addition, GC- rich stretches are not compatible with some synthetic nucleic acid chemistries
utilized in vector-based expression.
3.3 The target accessibility evaluation
Several studies have been done to illustrate the structural and sequence features affection
siRNA functionality, all of these aspects affect siRNA and mRNA accessibility (Patzel et al.
2005; Ladunga 2007). Target accessibility evaluation is crucial for proper designing of
efficient siRNA, as mRNA tends to form secondary structure that affects its accessibility and
hence reduces the capability to design siRNA targeting certain regions of mRNA. Therefore,

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target accessibility evaluation represents where the mRNA is more likely be accessed by
short oligomers as siRNAs, it involves not only mRNA secondary structure evaluation, but
also energetic calculation of siRNA and mRNA. For interaction between two RNA
sequences (siRNA and mRNA) two types of energies are needed: first energy required for
opening the binding site, second energy required to gain hybridization the summation of
these three energies is defined as interaction energy. The energy required for opening
siRNA duplex and mRNA should have lesser than the hybridization energy between siRNA
and the mRNA. There are evidence based correlation between siRNA inhibition efficiency
and siRNA-mRNA binding energy (Mckstein et al. 2006), that strengthens the findings of
Ladunga in which target accessibility information was found to provide the most predictive
feature among the 142 features studied and improves the prediction of highly efficient
siRNA (Ladunga 2007). Other parameters affecting target accessibility are presented below:
3.3.1 GC content
GC content represent the percentage of Guanine and Cytosine (two of the four nucleotides
types that build the mRNA) should not be too high in order not to impair the double strand
siRNA unwinding and enables the ease of RISC protein entrance.
3.3.2 Palindrome
Palindrome should be addressed in target accessibility evaluationwhere region(s) in one
strand binds to another region in the same strand due to reverse complementation.
Therefore, palindromes should be avoided in siRNA design as they tend to make intra-
molecular structure (2ry structure) which impairs RISC binding [Fig 6].

Fig. 6. Palindrome patterns and their affect on siRNA binding to RISC and the targeted
mRNA. Palindromes lead to changing double stranded siRNA secondary structure which in
turn affects their ability to bind to RISC and the targeted mRNA (Mysara 2010).


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3.3.3 Thermodynamic stability
It is important to keep/introduce relative thermodynamic stability at both ends of the
siRNA (3UTR and 5UTR) and low stability at the central zone as these facilitate ds-siRNA
cleavage.
3.3.4 Differential end stability
Differential end stability is considered one of the most important features that affect siRNA
functionality (Schwarz et al. 2003) [Fig 7]. RISC binds to either sense or the antisense strand,
but with different ratio. This ratio depends on Differential stability between the first
couple of bases ofthe 5 end from both strands. As these couple of bases affect what is called
Thermo-Dynamic stability (TDS), so the lower the stability the better it binds to RISC. It has
been found that only the antisense (leading) strand is capable of causing gene silencing
(Dorsett & Thomas Tuschl 2004). Therefore, it is essential design siRNA with low TDS at 5
end of the antisense than TDS of the 3 end, to have a better binding with RISC and better
efficiency in silencing the target mRNA.

Fig. 7. RISC and Differential end stability. This figure illustrates the effect of differential end
stability of RISC annealing with ds siRNA. Therefore, it is very important to ensure the 5
end of the siRNA lead-strand is less stable than the 5 end of the passenger-strand. This way
RISC would form complex with only the lead-strand that is designed to bind to the targeted
mRNA.
3.3.5 The number of single-stranded base pairs at the 5 and 3 ends of the target
mRNA
It has recently been shown to significantly contribute to the effectiveness of siRNAs by
Patzel and Kaufman in their recent (S. H. E. Kaufmann & Patzel 2008). (Patzel, Rutz et al.
2005) (Patzel, Rutz et al. 2005) (Patzel, Rutz et al. 2005) (Patzel, Rutz et al. 2005) The same
conclusion was reached by Gredell and co-workers in July 2008.
3.4 siRNA specificity [The off-targeting effect]
Ideally, the siRNA must not cause any effects other than those related to the knock down of the
target gene (Semizarov et al. 2003). It is essential that the designed siRNA affects only the

521
targeted mRNA. In other words, siRNA should not invoke innate immunity nor has any
off-targeted mRNA.
3.4.1 Innate immunity effect
Concerning the innate immunity effect, by rational selection of appropriate length of the
siRNA (21-23 nucleotides) the innate immunity will not be triggered (Birmingham et al.
2006). Although, duplexes of less than 30 nt are short enough to evade immunorecognition
by cytosolic double-stranded RNA (dsRNA) receptors, but are long enough to trigger Toll-
like receptor 7 sequence-dependent recognition (Patzel et al. 2005). Recognition of motifs as
5-GUCCUUCAA-3, 5-UGUGU-3 and tetrad-forming poly(G) stretches and avoidance of
their presence in the sensitized siRNA, help over coming Toll-like receptor recognition.
There was several works using chemical modification in order to mask the innate immunity
response (interferon response) (Patzel 2007).
3.4.2 Off-target effect
Apart from that, comes the problem of off-targets which is one of the most important factors
for siRNA selection and filtration. siRNA off-target is mainly any target that is affected by
siRNA other than the assigned target. It is very common for siRNA to have a multi-target as
they are only 21-23 nucleotide length; therefore, there is a good chance siRNA could match
with more than one mRNA. In fact, as observed in the work of Jackson et al, both sense and
antisense and know to have an off-target effect with several mRNA transcripts (Jackson et
al. 2003; Jackson & Linsley 2010). There are different mechanisms through which siRNA can
trigger off-targeting actions:
I) Complete or near complete off-target matches(siRNA-like effects)
This mechanism is triggered whenever the designed siRNA is completely identical (or with
one mismatch) with a region in the off-targeted mRNA. This complete (near complete)
matches between siRNA and mRNA leads to the destruction of that mRNA with the same
mechanism that siRNA silences the targeted mRNA as described before.
II) Partial off-target (miRNA-like effects through Seed matching off-target)
If the designed siRNA seeding region (second to seventh position) matches with 3UTR of
off-targeted mRNA, this will result in affecting the off-target translation as illustrated by (E.
M. Anderson et al. 2008). Therefore, these siRNAs are considered as partial off-targets and
should be excluded. Chemical modifications have been applied here to reduce off-target and
increase the specificity (Birmingham et al. 2007). These off-target effects (complete, near
complete or partial) are responsible for loss of specificity as they make this unwanted
silencing with other proteins synthesising genes. Moreover, they also cause loss of siRNA
potency as the unwanted off-targeting of other mRNA could lead to unavailability of these
siRNAs at the original targets (Semizarov et al. 2003; Vert et al. 2006).
In addition to those types of off-target effects, there is the protein interaction. As siRNAs are
known to bind to different cellular proteins and alter them, which is known as Aptamer
Effect as described in (Semizarov et al. 2003). Moreover, avoidance of sequence motifs
interfering with RNA synthesis and purification should be considered, as Guanine-rich
RNA sequences and sequences containing consecutive stretches of more than three G bases
(Patzel et al. 2005).


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3.5 siRNA duplex chemical modification
Several chemical modifications could be introduced to the designed siRNA in the aim of
enhancing its tolerability, improving it stability, limiting its off-target effect and conjugation
with tracking agent properly. There are multiple types of chemical modifications that are
typically introduced into siRNAs, as summarized in the work of (Birmingham et al. 2007):
I) Sense strand disabling: it is done to increase the specificity and efficiency of siRNA
designed. Various approaches were used as2 ribose modifications including 2-OR where
R fluoro, alkyl, O-alkyl4043; LNA modifications at the 5 end of the sense strand.
II) Stabilization: Chemical modifications of the phosphate backbone (e.g. phosphorothioate
linkages), the ribose (e.g. locked nucleic acids, 2-deoxy-2-uorouridine, 2-O-ethyl),and/or
the base (e.g.2-uoropyrimidines) increase the resistance of siRNA to nuclease. Stability of
siRNAs in biological fluids needs various modifications as 2-halogen, 2-alkyl and/or 2-O-
alkyl modifications of one or both strands of the siRNA as well as stabilizing internucleotide
modifications of the overhangs. Care should be taken when addressing those modifications
not to interfere with siRNA efficiency.
III) Specificity: Chemical modifications in the aim of increasing siRNA specificity and
decrease its off-target activity, include includes 2-O-alkyl modification of unique positions
of the sense and/or antisense strand. These modification patterns severely limit sense and
antisense off-target effects by disrupting seed-mediated off-target activity.
IV) Conjugations: siRNAs have been conjugated with lipophilic derivatives of cholesterol,
lauric acid or lithocholic acid to enhance their cellular uptake and specificity (Lorenz et al.
2004). The safest sites for conjugation are the 5 and 3 termini of the sense strand.
4. Guidelines for siRNA rational design
After have discussed factors influencing the siRNA efficacy,here we present our methodology
and phases for efficient siRNA rational design. Originally, this methodology was inspired by
the repeated Influenza pandemics, and our trials to design a novel siRNA therapy that would
work for any new pandemic(ElHefnawi, Alaidi et al. 2011). There are seven phases that should
be considered for proper designing of siRNA with high specificity and efficiency [Fig 8].
4.1 Targeted gene selection
Targeted gene selection is extremely critical for siRNA design as the purpose of gene
silencing is to stop the expression of specific abnormal proteins most commonly be involved in
the biological pathways as cancer pathways. Therefore, the protein of interest should play a
key role in this pathway, in order to produce the desired therapeutic effect from the silencing
process. Therefore, there is a need to search the key regularity protein annotated in various
biological pathway databases as Reactome and KEGG (http://www.reactome.org/ &
http://www.genome.jp/kegg/pathway.html) and design siRNA capable of targeting them.
4.2 Targeted sequence specification and filtration
After selecting the gene of interest, as gene itself is not targeted but rather its transcript(s),
all the available transcripts should be located. In some instance only one transcript should
be targeted; in this case all other transcripts should be excluded as targeting them is
considered as lack of specificity (off-target). But on the other hand, if there is a need to
silence all of the gene's transcripts (which is very common), several options are available for
handling such situation [Fig 9] either by mapping the transcripts on the genome, as

523

Fig. 8. Different phases for designing siRNA with high efficiency & sensitivity. There are
seven distinguished phases for siRNA design: 1st choosing the targeted gene for silencing.
2nd identifying the proper target sequence space that represent all genes transcripts and
doesnt have any unstable regions. 3rd designing all possible siRNA with nineteen
nucleotides length with both sense and antisense strand.4th these potential siRNAs are
scored and evaluated according to several scoring mechanisms and criteria and then filter
them according to produced scores. 5th siRNA are filtered according to target accessibility.
6th off-target filtration of the remaining siRNA is performed excluding siRNAs with
unwanted off-target effect. 8th select the best designed siRNAs that pass all the previous
filtration phases and achieve the highest predicted efficiency (Mysara 2010).


524
proposed in the work of (Y.-kyu Park et al. 2008), or via using multiple sequence alignment
(MSA). Multiple sequence alignment is performed either by aligning different gene
transcripts and selecting the transcript with the highest identity to the alignment profile, in
other word; select the transcript that is more capable of representing all the other transcripts.
Another manoeuvre is by considering all transcripts regions in common (conserved).

Fig. 9. Different approaches of handling multiple gene transcripts. There are two proposed
approaches 1st by aligning between different gene transcripts in order to get the alignment
profile and choose the closest transcript to the alignment profile. 2nd way is to get the gaped
consensus between these transcripts and choose the regions that are 100% conserved
between them (Mysara 2010).
The latter method ensures designing siRNA targeting all the genes transcripts, this is very
important as one mismatch between the target mRNA (transcript) and siRNA could
dramatically affect siRNA efficiency (Czauderna et al. 2003) there was noticeable decrease in
the efficiency of designed siRNA when induced central single nucleotide variation between
the siRNA and targeted mRNA, all together with the findings in the following works (M.
Amarzguioui 2003; Sayda M Elbashir et al. 2002). However, one of the main disadvantages
of this approach is that it narrows the target sequence space and it is possible that no active
siRNA will pass the multi-filtration phases described in [Fig 8]. In this occassion,sequence
space should be widened via inclusion of (3UTR and 5UTR) or using the first approach.
After locating the targeted sequence space, both SNPs and unstable (highly variable)
positions should be identified (if any) and any designed siRNAs targeting these
residues/regions should be rejected. This way the targeted sequence space will be limited to
mRNA (or the conserved region among different gene transcripts) either representing the
ORF or (ORF + 3UTR + 5UTR) free from any SNPs or unstable regions.
4.3 Designing all possible siRNA targeting the selected regions
This section illustrates the proper siRNA length that ensures high efficiency and stability
having neutral effect on host innate immunity. Then it discusses how to select siRNA from
the mRNA sequence space.

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4.3.1 Selection of the appropriate siRNA length
Using siRNA (with its short length) has better advantages over using long double stranded
RNAi as they do not trigger immune response and they also silence the targeted mRNA more
efficiently. However, siRNA with length equal to thirty nucleotides were found to be inactive
(S M Elbashir, Lendeckel, et al. 2001). After that the selection of proper siRNA length was
heavily studied, upper and lower limits have been assigned. It was found that shortening the
length from nineteen to seventeen affected the siRNA capability to silence the targeted gene; as
at least nineteen nucleotides are required for RISC binding. (Czauderna et al. 2003). To
establish the upper length limit, it was found that siRNAs with length from (18 to 23) are at
least eight folds more effective than other lengths. In addition the 24-25 nucleotide length
siRNAs were completely inactive (S M Elbashir, J Martinez, et al. 2001). Therefore, siRNA with
length 19-21 plus a 2-nt overhang is the appropriate length for siRNA design and any further
deviation above or below this length threshold will have a direct effect on the siRNA activity.
It was also demonstrated that using 2-3mer nucleotides dT 3' UTR overhangs increases the
efficiency of antisense strand loading to the RISC complex.
4.3.2 Picking up siRNA from sequence space
After establishing the desired siRNA length (19 nucleotides), all possible siRNA molecules
should be considered using one nucleotide shift per time [Fig 10] till reaching the end of
sequence space. Although the ideal case is that each gene would have only one transcript with
no SNPs nor highly variable regions, the vast majority of the genes sequence space would be
separate pieces (not intact) as shown in [Fig. 9]. Therefore, the selection of the 19 nucleotide
length siRNA should only be restricted to those sequence spaces free from any gaps.

Fig. 10. Designing of all possible siRNA using fixed frame shift. After choosing the
appropriate length (most propably 19 nucleotides + 2 overhangs) the target sequence space
is scanced and all possible siRNAs with one nucleotide shift at a time(Mysara 2010).


526
4.4 siRNA scoring and scores filtration
This stage is the most important stage for siRNA design as proper scoring and evaluation of
siRNA activity assist the time and cost consumtion. Moreover, developing siRNA scoring
tools with enhanced specificity and sensitivity would also serve a lot in that regardAs
normally single mRNA would produce thousand potential siRNAs, these siRNAs need to be
evaluated in order to filter them to smaller number suitable for experimental testing. There
are several tools have been developed to predict siRNA activity; these tools differ a lot in
these prediction capabilities in the terms of specificity and sensitivity. They use several rules
and trained with various datasets, therefore, careful evaluation and picking up the right tool
is essential for proper siRNA scoring phase. The details regarding siRNA scoring is further
explained in the next section of the chapter.
4.5 siRNAs target accessibility filtration
For interaction between two RNA sequences (siRNA and mRNA) two types of energies are
needed: first energy required for opening the binding site, second energy required to gain
hybridization. There are several programs that is used to calculate each energy among them
RNAduplex is capable of calculating duplex energy and RNAplfold capable of calculating
opening energy for ds-siRNA and targeted mRNA (target site accessibility energy).
Both RNAduplex and RNAplfold belong to Vienna RNA package
http://www.tbi.univie.ac.at/~ivo/ RNA/. There are two more tools that are able to provide
better advantages, RNAup and RNAxs.
4.5.1 RNAup
RNAup (that also belongs to Vienna RNA package) is capable of calculating all the three
energies required for assisting the interaction energy (Mckstein et al. 2006). RNAup starts
with calculating the probability that the sequence intervals (after splicing the sequence in
small subsequences) are unpaired. Then, it computes the interaction energy, ending with
choosing the ones with the least free energy (i.e. the highest stability). However, it cannot
handle sequences longer than 5000 nucleotides as it needs a lot of memory.
4.5.2 RNAxs
RNAxs program (modification of the older RNAplfold) is one of the programs used to
evaluate siRNA efficiency according to target accessibility evaluation; it combines
RNAplfold, RNAfold and RNAduplex (Tafer et al. 2008). RNAxs is able to provide two major
advantages: Time reduction and single phased process, which has been shown in the work
of (Hofacker & Tafer 2010) the comparative experiment done between RNAxs and 2 other
target accessibility based programs (OligoWalk & Sirna). It was found that only RNAxs is
able to identify siRNAs with inhibition efficiency >50% and to classify 50% of experiment
siRNA producing prediction capability higher than the other two programs.
4.6 siRNAs off-target filtration
All the siRNAs that pass the assigned thresholds for each scoring tools are filtered by their
tendency to trigger off-target effect. As described in section later (under siRNA specificity),
first siRNA having complete matches (or near complete) with the off-target mRNA should
be excluded (19/19 or 18/19 or 18/18). Next, the rest of the siRNAs are filtered according to

527
the presence of partial off-target by excluding siRNAs with matches between their seeding
regions (second to seventh position) and the 3 UTR of the off-targeted mRNAs. This way
the selected siRNA candidates would have the required specificity [Fig 11].

Fig. 11. Off-target filtration workflow describing decision making process for siRNAs off-
target filtration.
4.7 Selecting the best designed siRNA
The final step is sorting the acceptable siRNAs candidates according to the predicted inhibition
score. Taking the top 10-50 (if applicable) and order them for synthesis by adding UU or dTdT
to the 3 ends. Final result is a double strand siRNAs containing leading strand and antisense
strand with two 3end overhangs. There are also several chemical modifications could be
applied to the ds-siRNA would serve in increasing the stability, efficiency or neutralizing the
immune response by various possible modifications (Birmingham et al. 2007).


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4.8 Automation of siRNA design
This stepwise approach for designing siRNAs with acceptable target accessibility properties,
passing predicted score, SNPS and off-target filtration could be automated using various
programs and tools. The extend of considering those steps varies from program to another
according to the used algorithm and the state of the art protocol available at its time, in our
previous work, we managed to develop MysiRNA-Designer, siRNA design tool that
implements all of the steps presented above [Table 1](Mysara, J. Garibaldi, et al. 2011).

Tools name
M
u
l
t
i
-
t
r
a
n
s
c
r
i
p
t
s

C
o
n
s
i
d
e
r
a
t
i
o
n

C
o
n
s
e
r
v
e
d

R
e
g
i
o
n

A
n
a
l
y
s
i
s

S
N
P
s

E
v
a
l
u
a
t
i
o
n

M
u
l
t
i
-

a
l
g
o
r
i
t
h
m
s

S
c
o
r
i
n
g

2
r
y

s
t
r
u
c
t
u
r
e

E
v
a
l
u
a
t
i
o
n

T
a
r
g
e
t

a
c
c
e
s
s
i
b
i
l
i
t
y

F
u
l
l

H
o
m
o
l
o
g
y

O
f
f
-
t
a
r
g
e
t

S
e
e
d

R
e
g
i
o
n

o
f
f
-
t
a
r
g
e
t

S
e
r
v
e
r

B
a
s
e
d

MysiRNA-Designer + + + + + + + + -
siDESIGN Center *1 + + + - - - + + +
Asi-Designer *2 + - + - + - + - +
RNAxs *3 - - - - + + - - +
siDRM *4 - - - - - - + + +
Table 1. Comparison between MysiRNA-Designer and several programs used for siRNA
full automation designing. This Comparison involves tools ability to perform alignment
between different transcripts, conserved regions consideration, all together with siRNA
candidate evaluation using several algorithms and target accessibility. siRNAs filtration by
the presence of Single Nucleotide Polymorphisms and off-targets (both full homology and
seed regions)(Mysara, J. Garibaldi, et al. 2011, submitted). *1 siDESIGN Center at
http://www.dharmacon.com/designcenter/DesignCenterPage.aspx. *2 Asi-Designer
available at http://sysbio.kribb.re.kr:8080/AsiDesigner/menuDesigner.jsf. *3 RNAxs
available at http://rna.tbi.univie.ac.at/cgi-bin/RNAxs. *4 siDRM available at
http://sidrm.biolead.org/index.php.
5. Models used for predicting siRNA activity
There are several methods for scoring and predicting designed siRNA activity, some of
them are more accurate than the others; however, they are classified into two groups
(Ichihara et al. 2007): (i) Huesken dataset non-dependant [first generation]. (ii)Huesken
dataset dependant [second generation]

529
5.1 Huesken dataset non dependant [First Generation]
These tool were developed to select the most efficient siRNAs, and they depend on
differential ends Thermodynamic stability measures, mRNA secondary structure and base
preferences specific position target uniqueness. Example of these rules: Reynolds (Reynolds
et al. 2004), Amarzguioui (Mohammed Amarzguioui & Prydz 2004), Takasaki (Takasaki et
al. 2004), Katoh (Katoh & Suzuki 2007), Ui-Tei (Ui-Tei et al. 2004), Hsieh (Hsieh et al. 2004).
However, these first generation scoring techniques have shown to have low accuracy, as up
to 65% of the siRNAs predicted as active (by these tools) failed to achieve 90% inhibition
when tested experimentally and up to 20% of them were false positive, as described by (Ren
et al. 2006). Therefore, there was a need for another approach that does not only take the
site-specific position into consideration but also implement data mining techniques to
interpret the experimentally obtained data.
5.2 Huesken dataset dependant [Second Generation]
This class has been developed mainly through experimental data observation, as the existence
of dataset with fully annotated experimentally siRNAs with their different efficiency enabling
sophisticated data mining handling of this data, was not available until the dataset of Novartis
that was introduced by Huesken (Huesken et al. 2006) and used for training of several scoring
tool as: Biopredsi (Huesken et al. 2006), DSIR (Vert et al. 2006), ThermoComposition21 (S.A.
Shabalina et al. 2006), i-Score (Ichihara et al. 2007) and Scales (Matveeva et al. 2007).
These scoring techniques predict siRNA efficiency more accurately than the older tools.
Although they use completely different algorithms to evaluate siRNA efficiency, they have
very close accuracy compared to the rest of the second generation algorithms, as described
by (Ichihara et al. 2007). As in the comparative study done in the Ichiharas work all the
second generation (except for Scales) and only Reynold and Katoh from the first generation
achieved 90% successful prediction. Moreover, sensitivity of the second generation
compared to Reynold and Karoh (which appear to have approximate accuracy) was at least
8 fold lower that the second generation sensitivity (this also supports Rens findings
mentioned earlier). Here, we handle the basicinformation of each member of this group of
tools and provide comparison between them [Table 2].
5.2.1 Biopred
In Biopred, artificial neural network (ANN) was trained using huge number of records
(2,182 training and 259 test), considering not only single nucleotide residue but certain
patterns (as di-nucleotides). This work is considered the start of the second generation
siRNA approaches and noticeable shift in the scoring accuracy. Although ANN used in this
work provided ambiguity to the module and prevented further development (due to the
complexity of the model), it was considered, at that time period, the best way to handle all
these different parameters. The server based Biopred model was later simulated and
released as Biopredsi excel-based tool together with i-Score, which is going to be illustrated
later on (Ichihara et al. 2007).
5.2.2 DSIR
In DSIR, they used the exact training and test data as Biopredsi but with simplified linear
regression model to give prediction based to two main sequence features and three main
parameters with Pearson Correlation coefficient = 0.67. The main sequence feature is A/U
presence at the first position of the 5 end guidance strand and the absence of Cytosine from


530
both positions seven and eleven. The main parameters that have been used to build the
model are: sprase21, spectra21, composition representation. These three parameters divide
the siRNA by different manners and calculate the total score of all of them providing a very
representative and interpretable method to evaluate a siRNA sequence, see Table 2.

Parameter Description
Position
dependant
consensus

As several positions has been identified to be conserved between
effective siRNA, so the are scored out of 11 for the presence of
desirable residues and out of 10 (in -VE charge) for the presence of
undisirable residue in specific position.

Dinuleotide
Content Index

As the occurrence of some dinucleotide combinations have exceeded
the random distribution, so by combining these unique pairs with the
level of effectiveness of these siRNA, it was found (or precisely
confirmed) the low frequency of G/C dinucleotide pairs accompany
high siRNA efficacy.
Thermodynamic
Profile & Free
Energy (G)
It was found that the difference between 5 and 3 in free energy (or
its oppose stability) especially at the last 2,3,4,5 from each side
plays a crucial role in not only distinguishing the sense and antisense
but in efficiency evaluation.
Table 2. Description of parameters considered by ThermoComposition (Mysara 2010).
5.2.3 Thermo composition
Here a small number of parameters have been used in to train neural network using 653
siRNA-records as a training set. These parameters have been carefully selected from 18
parameters leading to this small number of parameters (three parameters), that had
provided the advantage of simplicity over other neural network as no need for huge
number of training dataset is required, and that opened the door for any further
development. The uniqueness in this work is that it combines the position dependant
features with Thermodynamic features [Table 2].
5.2.4 i-Score
In i-Score, linear regression model was built on identifying the nucleotide that is preferred
in each position and calculated the inhibition score (i-score) working on Huesken dataset
(2431) with Pearson correlation coefficient = 0.635. Also in this work they pointed out a very
important threshold as the exclusion of Thermostable siRNA (with stacking energy (whole
G) < -34.6 k.cal) improved the score accuracy of not only i-Score but also DSIR, Biopredsi
and ThermoComposition21 (Ichihara et al. 2007).

531
5.2.5 Scales
Linear regression model fitting with local stability of siRNA duplex and other parameters
was the way Matveevas team managed to score siRNA in siRNA Scales, using Huesken
dataset for training and three other dataset from various pharmaceutical companies for
validation. The use of linear regression provided additional advantages over neural
network, as it enabled the introduction of relevant importance to the same parameter at
different positions which cannot be applied to the same node parameter in the neural
network. In scales the linear regression was build on two sets of parameters: the first
group covers the stability of siRNA ends especially the 1st and last two base pair of the
siRNA the second group depends on evaluation of certain nucleotide at specific positions. A
comparison between all second generation tools is provided in [Table 3] (Mysara 2010).
However, all these models have limitations in performance. There are recent efforts to
enhance the siRNA scoring functionality through applying a second artificial intelligent
layer that depends on the predicted scores of other second generation tool, as in MysiRNA
model. It is siRNA functionality/efficacy prediction model that was developed by
combining two existing scoring algorithms (ThermoComposition21 and i-Score), together
with the whole stacking energy (G), in a multi-layer artificial neural network. It was found
that this kind of combination increases the correlation coefficient of the prediction accuracy
from (0.5 to 0.6) between scales and MysiRNA models (Mysara, M. Elhefnawi, et al. 2011,
submitted).
6. Experimental section
Here we present an example about working with the previously mentioned protocol for
proper siRNA design for targeting human TP53 gene that has been identified as oncogenes.
We start with finding P53 mRNA, by searching the NCBI Nucleotide dataset; we will find
mRNA refseq id NM_000546.4 for Homo sapiens tumor protein p53 (TP53), transcript
variant 1. Knowing that we need to target all the genes transcripts, we should find all
available transcripts. One way to do that is by blasting the mRNA refseq database, searching
for mRNA sharing the same name and organism, using NCBI remote Blast. Seven different
mRNAs were identified as following: NM_000546, NM_001126112, NM_001126115,
NM_001126117, NM_001126116, NM_001126114, NM_001126113. All of these transcripts
were later alignment together, as an approach to identify conserved regions. We used
ClustalW to align those 7 transcripts with their different lengths 2586, 2583, 2271, 2331, 2404,
2719 and 2646 respectively. The resulted alignment file, was the treated with cons tool to
find the consensus between those transcripts, using 100% conservation.

Tools Model
Technique
Training
Dataset used
Tool
available at
Disadvantages
Biopredsi (Huesken
et al. 2006)
Neural
network
2,431 records
from Huesken
dataset.
http://www.bi
opredsi.org

Possible over
estimation due
to over fitting
of training set
with test (S.A.
Shabalina et al.
2006)


532
Tools Model
Technique
Training
Dataset used
Tool
available at
Disadvantages
DSIR (Vert et al.
2006)
Linear
Regression
2,431 records
from Huesken
dataset.
http://biodev.
extra.cea.fr/DS
IR/DSIR.html
Too many
parameters
resulted in
slow
performance
ThermoCompsition*
(S.A. Shabalina et al.
2006)
Neural
network
653 records from
different
publications.
ftp://ftp.ncbi.
nlm.nih.gov/p
ub/shabalin/si
RNA/Thermo
Composition
Small number
of parameters
for ANN
training. Slow
due to
secondary
structure
calculation
i-Score (Ichihara et
al. 2007)
Linear
Regression
2,431 records
from Huesken
dataset.
http://www.
med.nagoya-
u.ac.jp/neurog
enetics/i_Score
/i_score.html
Relatively low
statistical
accuracy
Scales (Matveeva et
al. 2007)
Linear
Regression
2,431 records
from Huesken
dataset.
http://gestela
nd.genetics.uta
h.edu/siRNA_
scales/
Relatively low
statistical
accuracy
Table 3. Comparison between different second generation scoring techniques (Mysara 2010).
*In case of ThermoComposition19, however, Huesken dataset used for training
ThermoComposition21 and 653 siRNA used for validation.
The resulted consensus was found as per below:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNCCANGNNATGGANGNTNNNNTGNNCTGTNNNCNNACNAN
NNTGNNCNANGNNTNANTGANAGANCNAGNNCCNNNNNNANNNNNNANAAN
NNNANAGNNNNNNNCNCCNGNGGNNNNNNCNCCANCNNNNNCTANNNCGNN
NGNNNNNTGCANNAGNNNCNTNCNNNNNNNTGTNNNNTTNCTGNCNNNTNCCA
GNNNNNNTANCNGNNCANNTNNGNNNTNNNTNTNNNCTNNNTGNNTNCTNNN
NNNNNNNNNTCNNNNNCNTNCANGTACTCCCCTGCCCTCAACAAGATGTTTTGCC
AACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGC
ACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGT
GAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCA
GCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACA

533
CTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTAC
CACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGA
GGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGG
AACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGA
AGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACT
AAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCAC
TGGATGGAGAATATTTCACCCTTCAGNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGC
CTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCAC
TCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCAT
GTTCAAGACAGAAGGGCCTGACTCAGACTGACATTCTCCACTTCTTGTTCCCCACTG
ACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTTGAAC
CCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCG
GGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAGGAGGATGGG
GAGTAGGACATACCAGCTTAGATTTTAAGGTTTTTACTGTGAGGGATGTTTGGGAGA
TGTAAGAAATGTTCTTGCAGTTAAGGGTTAGTTTACAATCAGCCACATTCTAGGTAG
GGGCCCACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTA
ACTTCAAGGCCCATATCTGTGAAATGCTGGCATTTGCACCTACCTCACAGAGTGCAT
TGTGAGGGTTAATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGG
GGTCTAGAACTTGACCCCCTTGAGGGTGCTTGTTCCCTCTCCCTGTTGGTCGGTGGGT
TGGTAGTTTCTACAGTTGGGCAGCTGGTTAGGTAGAGGGAGTTGTCAAGTCTCTGCT
GGCCCAGCCAAACCCTGTCTGACAACCTCTTGGTGAACCTTAGTACCTAAAAGGAA
ATCTCACCCCATCCCACACCCTGGAGGATTTCATCTCTTGTATATGATGATCTGGATC
CACCAAGACTTGTTTTATGCTCAGGGTCAATTTCTTTTTTCTTTTTTTTTTTTTTTTTTCT
TTTTCTTTGAGACTGGGTCTCGCTTTGTTGCCCAGGCTGGAGTGGAGTGGCGTGATCT
TGGCTTACTGCAGCCTTTGCCTCCCCGGCTCGAGCAGTCCTGCCTCAGCCTCCGGAG
TAGCTGGGACCACAGGTTCATGCCACCATGGCCAGCCAACTTTTGCATGTTTTGTAG
AGATGGGGTCTCACAGTGTTGCCCAGGCTGGTCTCAAACTCCTGGGCTCAGGCGATC
CACCTGTCTCAGCCTCCCAGAGTGCTGGGATTACAATTGTGAGCCACCACGTCCAGC
TGGAAGGGTCAACATCTTTTACATTCTGCAAGCACATCTGCATTTTCACCCCACCCTT
CCCCTCCTTCTCCCTTTTTATATCCCATTTTTATATCGATCTCTTATTTTACAATAAAA
CTTTGCTGCCACCTGTGTGTCTGAGGGGTG
Then, we used this consensus to evaluate its target accessibility using RNAxs, finding all
possible regions to be targeted by siRNA. 1033 possible siRNA were designed using RNAxs.
Those siRNAs were evaluated using 10 siRNA efficiency prediction tools as Reynolds
(Reynolds et al. 2004), Amarzguioui (Mohammed Amarzguioui & Prydz 2004), Takasaki
(Takasaki et al. 2004), Katoh (Katoh & Suzuki 2007), Ui-Tei (Ui-Tei et al. 2004), Hsieh (Hsieh
et al. 2004), Biopredsi (Huesken et al. 2006), DSIR (Vert et al. 2006), ThermoComposition21 (S.A.
Shabalina et al. 2006) and i-Score (Ichihara et al. 2007). Selecting siRNA passing 90% or 0.90
predicted score. 111 siRNAs passed these filtration processes, those siRNAs were searched
to identify SNPs occurrence residues. All of those 111 siRNAs were found to be targeting
SNPs free regions. The last step was to filter those siRNAs against mRNA dataset, to
identify those having off-targets. Any siRNA with either complete or partial off-target
should be excluded. 85 siRNAs were found to be off-target free candidates. Finally they
were filtered and only siRNA with inhibition efficiency above 90%, according to MysiRNA
model, were accepted.


534
siRNA
position
Sense Antisense
Predicted
efficiency
800 GCGUGUGGAGUAUUUGGAU AUCCAAAUACUCCACACGCaa 90.6%
822 AGAAACACUUUUCGACAUA UAUGUCGAAAAGUGUUUCUgu 92.6%
883 GUACCACCAUCCACUACAA UUGUAGUGGAUGGUGGUACag 91.5%
1330 CCCGCCAUAAAAAACUCAU AUGAGUUUUUUAUGGCGGGag 91.4%
1842 GAAACCACCUUUUAUUACA UGUAAUAAAAGGUGGUUUCaa 92.3%
1915 GGUGGGUUGGUAGUUUCUA UAGAAACUACCAACCCACCga 92.1%
1919 GGUUGGUAGUUUCUACAGU ACUGUAGAAACUACCAACCca 90%
2016 CCUUAGUACCUAAAAGGAA UUCCUUUUAGGUACUAAGGuu 95.4%
2111 GCUCAGGGUCAAUUUCUUU AAAGAAAUUGACCCUGAGCau 92.9%
2499 CCCUCCUUCUCCCUUUUUA UAAAAAGGGAGAAGGAGGGga 92%
2530 CUCCCUUUUUAUAUCCCAU AUGGGAUAUAAAAAGGGAGaa 91.5%
25030 AUAUCGAUCUCUUAUUUUA UAAAAUAAGAGAUCGAUAUaa 93.1%
Table 4. Final siRNA candidates after all stages of design and filtration.
In ElHefnawi et. Al., other examples of optimal siRNA design and selection as silencers for
difficult targets such as the Hepatitis C virus (HCV), and the Influenza a virus that have
been experimentally tested for verifications of the methodology are under publication
(Mahmoud ElHefnawi1 2011) (Mahmoud ElHefnawi 1 2011)).
7. Conclusion
In this chapter we provide a comprehensive foundation of the underlying bioinformatics
methodology for optimal design and selection of siRNA molecules. We address factors
affecting siRNA interference, covering both siRNA and mRNA sides. These factors can be
classified into four major classes, the first class of factors, targeted region or target
sequence space, addresses how to identify regions in the mRNA that should be targeted by
the designed siRNA; and discusses five factor affecting target sequence space: transcript
region, transcript size, mRNA multiple splicing, single nucleotide polymorphism and
orthologs consensus. The second class of factors, siRNA sequence space, addresses
positional/word preferences in the sense/antisense strand of the siRNA. siRNA sequence
space is affected by several factors including nucleotide positional preferences Protocol, GC
content, and palindrome. In addition, thermodynamic stability and differential ends
instability have been identified to be highly important factors in siRNA functionality. The
third class of factors, is the target accessibility, and how the targeted mRNAs tend to
form secondary structure that affect their accessibility hence reduce the capabilities of the
designed siRNA to target certain regions of mRNA. Target accessibility is considered as the
sum of the energy required to open mRNA and siRNA duplex and the energy required to
stabilize siRNA-mRNA duplex. The fourth class of factors, off-target matches, that
influence siRNA specificity via perfect-match, and partial off targets & sequence motifs that
invoke immune reaction. Each of these classes can greatly affect siRNA selection and
therefore are studied thoroughly in this chapter.
We present a step wise protocol for designing siRNA with the highest specificity and
sensitivity in seven different phases, Targeted gene assignment, targeted sequence
specification and filtration, designing all possible siRNAs targeting the selected regions,
siRNAs scoring and scores filtration, siRNAs target accessibility filtration, siRNAs off-target

535
filtration, selecting the best designed siRNA. We cover state of the art tools for siRNA
efficiency prediction, in two generation: the first generation tools select the most efficient
siRNAs depending on differential ends thermodynamic stability measures, mRNA
secondary structure and base preferences specific position target uniqueness. The second
generation tools have been developed by applying sophisticated data mining techniques to
handle huge annotated records of siRNAs with their experimental inhibition, as in Biopredsi,
ThermoComposition21 and Scaless artificial neural network model and DSIR and i-Scores
linear regression model. By the end of the chapter, we design siRNA targeting human P53
protein, as a practical example of the proposed protocol. Future directions would be to find
additional factors that affect shRNA (siRNAs inserted into expression vectors) that further
decrease the efficacy of the expressed siRNAs from them, and extending this methodology
latter for miRNA target recognitionpredictions.
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537
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across most Influenza A viral strains." Virol J 8: 44.
BACKGROUND: Influenza A virus poses a continuous threat to global public
health. Design of novel universal drugs and vaccine requires a careful analysis of
different strains of Influenza A viral genome from diverse hosts and subtypes. We
performed a systematic in silico analysis of Influenza A viral segments of all
available Influenza A viral strains and subtypes and grouped them based on host,
subtype, and years isolated, and through multiple sequence alignments we
extrapolated conserved regions, motifs, and accessible regions for functional
mapping and annotation. RESULTS: Across all species and strains 87 highly
conserved regions (conservation percentage > = 90%) and 19 functional motifs
(conservation percentage = 100%) were found in PB2, PB1, PA, NP, M, and NS
segments. The conservation percentage of these segments ranged between 94-98%
in human strains (the most conserved), 85-93% in swine strains (the most variable),
and 91-94% in avian strains. The most conserved segment was different in each
host (PB1 for human strains, NS for avian strains, and M for swine strains). Target
accessibility prediction yielded 324 accessible regions, with a single stranded
probability > 0.5, of which 78 coincided with conserved regions. Some of the
interesting annotations in these regions included sites for protein-protein
interactions, the RNA binding groove, and the proton ion channel.
CONCLUSIONS: The influenza virus has evolved to adapt to its host through
variations in the GC content and conservation percentage of the conserved regions.
Nineteen universal conserved functional motifs were discovered, of which some
were accessible regions with interesting biological functions. These regions will
serve as a foundation for universal drug targets as well as universal vaccine design.
Mahmoud ElHefnawi1, Rania Siam3, Nafisa Hassan2 , Mona Kamar2 , Marco Sgarbanti4,
Annalisa Rimoli4, Iman El-Azab5, Osama AlAidy6, Giulia Marsiliin Marco
Sgarbanti4 (2011). "The design of optimal therapeutic small interfering RNA
molecules targeting diverse strains of influenza A virus." Bioinformatics OUP under
revision.


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Mahmoud ElHefnawi 1, TaeKyu Kim3, Mona A. Kamar 2, Nafisa M. Hassan 2, Iman A El-
Azab4, Suher Zada5, Marc P. Windisch3* (2011). "Novel DESIGN AND
SELECTION OF EFFICIENT SPECIFIC UNIVERSAL SMALL INTERFERING RNA
MOLECULES tested in Hepatitis C Virus replicon cell lines." PLOS1 submitted.
Patzel, V., S. Rutz, et al. (2005). "Design of siRNAs producing unstructured guide-RNAs
results in improved RNA interference efficiency." Nat Biotechnol 23(11): 1440-1444.
24
MicroRNA Targeting in Heart:
A Theoretical Analysis
Zhiguo Wang
Research Centre, Montreal Heart Institute, Montreal, and Department of Medicine,
Universite de Montreal, Montreal,
Canada
1. Introduction
Cardiovascular disease remains the major cause of morbidity and mortality; according to
statistics, heart failure, the syndrome consequential to many diseases of the cardiovascular
system, is estimated to have a prevalence of 12% and an annual incidence of 510 per 1,000
in the developed countries and is the leading cause of hospitalization in the population over
50 years of age. Worse so, there is a clear tendency of increasing prevalence of
cardiovascular disease in this planet particularly in the developing nations. This problem
casts enormous health concern and costly socioeconomic burden worldwide.
We have entered post-genome era after the human genome project had been completed
years ago. Only <2% of all transcribed bases of the entire human genome constitutes the
genetic sequence encoding proteins and the rest of 98% accounting for ~70% of all genes
carry the sequences for RNAs not encoding a polypeptide chain that was used to be
considered for many years junk DNA of no physiologic function; proteins were generally
assumed the sole biopolymer capable of regulatory function. Intriguinly, the proportion of
transcribed non-protein-coding sequences increases with developmental complexity and is a
better indicator of phylogenetic level than the number of protein-coding genes of an
organism. It is now known that junk DNA encodes non-protein-coding RNAs (ncRNAs)
that are involved in determining the expression of protein-coding genes by regulating the
activity of that 2% of the genome. These ncRNAs include microRNAs (miRNAs), once
ignored completely or overlooked as cellular detritus, which were discovered over a decade
ago have recently taken many by surprise because of their widespread expression and
diverse functions.
miRNAs are endogenous small mRNAs of ~22 nucleotide in length which act primarily to
repress gene expression at the post-transcriptional level. To date, ~6400 vertebrates mature
miRNAs have been registered in miRBase, an online repository for miRNA, among which
~5100 miRNAs are found in mammals which include >850 human miRNAs. These miRNAs
are predicted to regulates >60% of proteincoding genes. The discovery of miRNA
challenges the central dogma of molecular biology that has be hold since the latter half of
the 20th century. With the recent rapid evolution of miRNA research, researchers have
begun to appreciate the roles of these small non-protein-coding mRNAs in the
cardiovascular system. Based on the published studies, it is now clear that miRNAs are
involved in nearly all aspects of cardiac function and pathogenesis (Wang, 2010; Wang et al.,
2008; Yang et al., 2008).

It known that an individual miRNA has the potential to target multiple protein-coding
genes and vice versa a single protein-coding gene may be regulated by multiple miRNAs,
implying that the action of miRNAs is not gene-specific. This fact creates an obstacle to our
thorough understanding of miRNA functions and the mechanism underlying these
functions. For example, we have recently found that miR-125-5p target GJA1 (encoding gap
junction channel protein connexin43) and GJC1 (encoding another gap junction channel
protein connexin40) to cause slowing of both ventricular and atrial conduction promoting
arrhythmogenesis in failing heart where its expression is upregulated (Wang, 2010).
However, based upon computational prediction it can also target SCN5A (encoding Nav1.5
Na
+
channel -subunit). An immediate question is whether the potential repression of
SCN5A also plays a role in miR-125-5p-induced conduction slowing. On the other hand,
GJA1, GJC1 and SCN5A are predicted to be regulated by other miRNAs in addition to miR-
125-5p (such as miR-101, miR-125, miR-130, miR-19, miR-23, miR-26 and miR-30); whether
these miRNAs are also involved in the deregulation of these genes in heart failure remained
unknown. This same uncertainty may exist in the interactions between literally all miRNAs
and protein-coding genes. Proper experimental approaches are ultimately required to clarify
these issues. However, at present it is not feasible to have thorough elucidation of the
complete set of target genes of a given miRNA or of the complete array of mRNAs that
regulate a given protein-coding genes; computational prediction remains the best alternative
for rapid identification of miRNA target genes.
This chapter aims to shed light on how to take rational uses of bioinformatics analysis to
identify the miRNAs from the currently available miRNA databases which have the
potential to regulate human genes related to cardiac function and pathology and to validate
the analysis taking several pathological settings associated with the deregulated miRNAs in
the heart. The pathological conditions to be discussed include arrhythmogenesis, apoptosis
and fibrogenesis that are known to be critical to the adverse electrical, cellular and structural
remodelling processes in various diseased states of heart such as myocardial infarction,
cardiac hypotrophy and heart failure.
2. Ion channel genes as targets for miRNAs
Cardiac cells are excitable cells that can generate and propagate excitations; At the cellular
level, excitability is reflected by cardiac action potential, which is orchestrated by
transmembrane proteins like ion channels and transporters and intracellular proteins for
Ca
2+
handling. (Wang, 2010; Wang et al., 2008; Yang et al., 2008). Deregulation of miRNA
expression has been implicated in a variety of diseased conditions of the heart and aberrant
expression of miRNAs can render expression deregulation of ion channel genes resulting in
channelopathiesarrhythmogenesis leading to sudden cardiac death. Indeed, we and others
have shown the critical involvement of miRNAs, particularly the muscle-specific miRNAs
miR-1 and miR-133, in regulating every aspects of cardiac excitability to affect
arrhythmogenesis under various pathological conditions including myocardial infarction,
cardiac hypertrophy, diabetic cardiomyopathy, etc. This property of miRNAs is conferred
by their ability to target ion channels and intracellular Ca
2+
handling proteins at the post-
transcriptional level, as already revealed by numerous studies. Even though, our current
knowledge about miRNA regulation of cardiac ion channels is still rather preliminary with
limited experimental data available in the literature. This is largely due to the limitations of

MicroRNA Targeting in Heart: A Theoretical Analysis 541
our technologies and approaches to conduct thorough characterization of miRNA targeting.
As a surrogate to these limitations, we have conducted a rationally designed bioinformatics
analysis in conjunction with experimental approaches to identify the miRNAs which have
the potential to regulate human cardiac ion channel genes and to validate the analysis with
several pathological settings associated with the deregulated miRNAs and ion channel
genes in the heart (Luo et al., 2010). In this way, we have been able to identify an array of
miRNAs that are expressed in cardiac cells and have the potential to regulate the genes
encoding cardiac ion channels, transporters and intracellular Ca
2+
handling proteins. Our
data well explain the ionic remodelling processes occurring in hypertrophy/heart failure,
myocardial ischemia, or atrial fibrillation at the level of miRNA; the changes of miRNAs
appear to have anti-correlation with the changes of many of the genes encoding cardiac ion
channels under these pathological conditions.
2.1 In Silico analysis of miRNA targets
We used the miRecords miRNA database and target-prediction website for our initial
analysis. The miRecords is resource for animal miRNA-target interactions developed at the
University of Minnesota (Xiao et al., 2009). The miRecords consists of two separate
databases. The Validated Targets database contains the experimentally validated miRNA
targets being updated from meticulous literature curation. The Predicted Targets database
of miRecords is an integration of predicted miRNA targets produced by 11 established
miRNA target prediction programs. These algorithms include DIANA-microT,
MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22,
RNAhybrid, and TargetScan/TargetScanS.
As an initial screening process, we performed miRNA target prediction through the
miRecords database (Xiao et al., 2009). This miRNA database integrates miRNA target
predictions by 11 algorithms. Four of the 11 algorithms (microInspector, miTarget,
NBmiRTar, and RNA22) were removed from our data analysis because they failed to
predict; these websites require manual input of 3UTR sequences of the genes. Thus, our
data analysis was based upon the prediction from seven algorithms (TargetScan, DIANA-
miT3.0, miRanda, PicTar, PITA, RNAHybrid, and miRTarget2) (Enright et al., 2003; Kertesz
et al., 2007; Kiriakidou et al., 2004; Krek et al., 2005; Lewis et al., 2003 & 2005; Rehmsmeier et
al., 2004; Wang & El Naqa., 2008). These prediction techniques are based on algorithms with
different parameters (such as miRNA seed:mRNA 3UTR complementarity, thermodynamic
stability of base-pairing (assessed by free energy), evolutionary conservation across
orthologous 3UTRs in multiple species, structural accessibility of the binding sites,
nucleotide composition beyond the seed sequence, number of binding sites in 3UTR, and
anti-correlation between miRNAs and their target mRNAs). A then-updated set of RefSeq
genes and their annotations was used to define a set of human 3' UTRs. Orthologous UTRs
(based on whole-genome alignments) were obtained for 22 other species from UCSC
Genome Bioinformatics. Conservation of each miRNA site was evaluated using
phylogenetic branch lengths of all species containing the site based on the methods by
Friedman et al (2009). For all highly conserved miRNAs, the probability of preferentially
conserved targeting for each site was estimated as described (Friedman et al., 2009).
Predicted consequential pairing to 3' end of miRNAs was included only if the raw 3' pairing
score (Grimson et al., 2007) is at least 3.0.


Fig. 1. Flow chart illustrating the procedures of our analysis. The First Dataset include all
miRNAs in the then-updated database that have the potential to target at least an ion-
channel-coding gene as predicted by at least 4 of the 7 algorithms. The Second Dataset limits
the miRNAs from the First Dataset to only those that are expressed in cardiac cells. These
miRNAs represent those that most likely play a role in regulating ion channel expression in
the heart. The Third Datasets are the lists of miRNAs that have been shown to be
deregulated in the pathological conditions under analysis, e.g. myocardial infarction, heart
failure, etc. These miRNAs represent those that likely play an important role in defining ion
channel expression under a given diseased state.
Each of the seven algorithms provides a unique dataset. Some of the algorithms have higher
sensitivity of prediction but lower accuracy and the others weight on the accuracy in the
face of reduced sensitivity. We collected all miRNAs predicted by at least four of the seven
algorithms to have the potential to target any one of the selected cardiac ion channel and ion
transporter genes. Meanwhile, we also collected all ion channel and ion transporter genes
that contain the potential target site(s) (the binding site(s) with favourable free energy
profiles) for at least one of then-registered 718 human miRNAs in the miRNA database
(miRBase).
Expression of miRNAs is tightly controlled by the genetic programme to ensure certain
spatial (depending on cell-, tissue-, or organ-type) and temporal (depending on
developmental stage) patterns. The expression profile under a defined condition is
considered the miRNA expression signature or miRNA transcriptome of a particular tissue.
One way to minimize the possibility of false positive predictions and to narrow down the
list of putative miRNA targets would be to compare the in silico target predictions to the
miRNA transcriptome signatures in the biological system of interest. We therefore
conducted miRNA microarray analysis of miRNAs including all 718 human miRNAs for
their expression in left ventricular tissues of five healthy human individuals. Using this set
of cardiac miRNA expression profiling data in conjunction with published data obtained by
real-time RT-PCR by Liang et al (2007), we refined the miRNAtarget prediction by filtering
out the miRNAs that are not expressed in the heart and focusing on the top 20 abundant

miRNAs in human heart (miR-1, miR-133a/b, miR-16, miR-100, miR-125a/b, miR-126, miR-145,
miR-195, miR-199*, miR-20a/b, miR-21, miR-26a/b, miR-24, miR-23, miR-29a/b, miR-27a/b, miR-
30a/b/c, miR-92a/b, miR-99, and let-7a/c/f/g). In this way, we generated the modified datasets
for subsequent analyses and obtained an overall picture of control of expression of ion
channel genes by miRNAs in heart under normal conditions.
Next, we intended to apply the theoretical prediction to explaining some established
observations of the electrical remodeling related to deregulation of both miRNAs and the
genes for ion channels and transporters. Three pathological conditions, cardiac
hypertrophy/heart failure, ischemic myocardial injuries, and atrial fibrillation, were studied
based on the expression profiles and participations of miRNAs in these conditions as
previously reported. The results are presented in the section following the next.
2.2 Control of expression of Ion channel genes by miRNAs under normal conditions
The above analyses allow us to reach the following notes.
1. One hundred ninety-three out of 718 human miRNAs or out of 222 miRNAs expressed
in the heart have the potential to target the genes encoding human cardiac ion channels
and transporters.
2. Only two genes CLCN2 and KCNE2 were predicted not to contain the target site for
miRNAs expressed in the heart.
3. It appears that the most fundamental and critical ion channels governing cardiac
excitability have the largest numbers of miRNAs as their regulators. These include
SCN5A for I
Na
(responsible for the upstroke of the cardiac action potential thereby the
conduction of excitations), CACNA1C/CACNB2 for I
Ca,L
(accounting for the
characteristic long plateau of the cardiac action potential and excitation-contraction
coupling), KCNJ2 for I
K1
(sets and maintains the cardiac membrane potential), SLC8A1
for NCX1 (an antiporter membrane protein which removes Ca
2+
from cells), GJA1/GJC1
(gap junction channel responsible for intercellular conduction of excitation), and
ATP1B1 for Na
+
/K
+
pump (establishing and maintaining the normal electrochemical
gradients of Na
+
and K
+
across the plasma membrane). Each of these genes is
theoretically regulated by >30 miRNAs.
4. The atrium-specific ion channels, including Kir3.4 for I
KACh
, Kv1.5 for I
Kur
, and
CACNA1G for I
Ca,T
, seem to be the rare targets for miRNAs (<5 miRNAs).
5. All four genes for K
+
channel auxiliary -subunits KCNE1, KCNE2, KCHiP, and
KCNAB2 were also found to have less number of regulator miRNAs (<10).
6. Intriguingly, 16 of these top 20 miRNAs are included in the list of the predicted
miRNA-target dataset; the other four cardiac-abundant miRNAs miR-21, miR-99, miR-
100 and miR-126 are predicted unable to regulate the genes for human cardiac ion
channels and transporters.
7. There is a rough correlation between the number of predicted targets and the abundance
of miRNAs in the heart. It appears that the miRNAs within top 8 separate from the rest 12
less abundant miRNAs in their number of target genes. The muscle-specific miRNA miR-
1 was predicted to have the largest number of target genes (9 genes) among all miRNAs
most abundantly expressed in the heart, followed by miR-30a/b/c, miR-24 and miR-
125a/b that have 6 target genes each. The muscle-specific miRNA miR-133 has four target
genes and three of them (KCNH2, KCNQ1 and HCN2) have been experimentally verified
(Luo et al., 2008; J Xiao et al., 2007; L Xiao et al., 2008).

8. Comparison of the target genes of the three muscle-specific miRNAs miR-1, miR-133
and miR-208 revealed that they might play different role in regulating cardiac
excitability. It appears that miR-1 may be involved in all different aspects of cardiac
excitability: cardiac conduction by targeting GJA1 and KCNJ2, cardiac automaticity by
targeting HCN2 and HCN4, cardiac repolarization by targeting KCNA5, KCND2 and
KCNE1, and Ca
2+
handling by targeting SLC8A1. By comparison, miR-133a/b mainly
controls cardiac repolarization through targeting KCNH2 (encoding HERG/I
Kr
) and
KCNQ1 (encoding KvLQT1/I
Ks
), the two major repolarizing K
+
channels in the heart.
miR-208 was predicted to target only KCNJ2 (encoding Kir2.1 for I
K1
). The non-muscle-
specific let-7 seed family members seem to regulate mainly cardiac conduction by
targeting SCN5A (Nav1.5 for intracellular conduction) and GJC1 (Cx45 for intercellular
conduction). miR-30a/b/c and miR-26a/b, miR-125a/b, miR-16, and miR-27a/b were
predicted to be L-type Ca
2+
channel blockers through repressing 1c- and/or 1/2-
subunits.
2.3 Application of our bioinformatics analysis to heart failure
The mechanisms for arrhythmogenesis in failing heart involve (Nattel et al., 2007): (1)
Abnormalities in spontaneous pacemaking function (enhanced cardiac automaticity) as a
result of increases in atrial and ventricular If due to increased expression of HCN4 channel
may contribute to ectopic beat formation in CHF; (2) Slowing of cardiac repolarization
thereby prolongation of APD due to reductions of repolarizing K
+
currents (including I
K1
,
I
Ks
, and I
to1
) provides the condition for occurrence of early afterdepolarizations (EADs)
leading to triggered activities; (3) Delayed afterdepolarizations (DADs) due to enhanced
Na
+
-Ca
2+
exchanger (NCX1) activity in cardiac hypertrophy/CHF is a consistent finding by
numerous studies. Upregulation of NCX1 expression is the major cause for the
enhancement; (4) Reentrant activity due to slowing of cardiac conduction velocity.
To date, there have been seven published studies on role of miRNAs and cardiac
hypertrophy (Car et al., 2007; Cheng et al., 2007; Sayed et al., 2008; Tatsuguch et al., 2007;
Thum et al., 2007; van Rooij et al., 2006, 2007). The common finding of these studies is that
an array of miRNAs is significantly altered in their expression, either up- or down-
regulated, and that single miRNAs can critically determine the generation and progression
of cardiac hypertrophy. The most consistent changes reported by these studies are up-
regulation of miR-21 (6 of 6 studies), miR-23a (4 of 6), miR-125b (5 of 6), miR-214 (4 of 6),
miR-24 (3 of 6), miR-29 (3 of 6) and miR-195 (3 of 6), and down-regulation of miR-1, miR-
133, miR-150 (5 of 6 studies) and miR-30 (5 of 6). These miRNAs were therefore included in
our analysis of target genes encoding ion channel and transporter proteins. Our analyses
suggest the following.
1. It is known that cardiac myocytes are characterized with re-expression of the funny
current (or pacemaker current) If that may underlie the increased risk of
arrhythmogenesis in hypertrophic and failing heart (Luo et al., 2008), which is carried
by HCN2 channel in cardiac muscles. We have previously verified that downregulation
of miR-1 and miR-133 caused upregulation of HCN2 in cardiac hypertrophy (Luo et al.,
2008). This may contribute to the enhanced abnormal cardiac automaticity and the
associated arrhythmias in CHF.
2. The NCX1 is upregulated in cardiac hypertrophy, ischemia, and failure. This
upregulation can have an effect on Ca
2+
transients and possibly contribute to diastolic

dysfunction and an increased risk of arrhythmias (Flesch et al., 1996; Nattel et al., 2007;
Pogwizd & Bers, 2002). Our target prediction indicates that SLC8A1, the gene encoding
NXC1 protein, is a potential target for both miR-1 and miR-30a/b/c. The
downregulation of miR-1 and miR-30a/b/c in hypertrophy/failure is deemed to relieve
the repression of SLC8A1/NCX1 since a strong tonic repression miR-1 and miR-
30a/b/c is anticipated considering the high abundance of these miRNAs. On the other
hand, upregulation of miR-214 tends to repress NCX1, but the expression level of miR-
214 is of no comparison with those of miR-1 and miR-30a/b/c; its offsetting effect
should be minimal. Our prediction thus provides a plausible explanation for the
upregulation of NCX1 through the miRNA mechanism.
3. A variety of Na
+
channel abnormalities have been demonstrated in heart failure. Several
studies suggest that peak I
Na
is reduced which can cause slowing of cardiac conduction
and promote re-entrant arrhythmias (Zicha et al., 2004). It has been speculated that
post-transcriptional reduction of the cardiac I
Na
-subunit protein Nav1.5 may account
for the reduction of peak I
Na
. In this study, we found that the only miRNA that can
target Nav1.5 and is upregulated in cardiac hypertrophy/CHF is miR-125a/b. As an
abundantly expressed miRNA, upregulation of miR-125a/b could well result in
repression of SCN5A/Nav1.5.
4. The gap junction channel proteins connexin43, connexin45 and connexin40 are
important for cell-to-cell propagation of excitations. Downregulation of connexin43
expression is associated with an increased likelihood of ventricular tachyarrhythmias in
heart failure (Kitamura et al., 2002). Other connexins, including connexin45 (Yamada et
al., 2003) and connexin40 (Dupont et al., 2001), are upregulated in failing hearts,
possibly as a compensation for connexin43 downregulation. Our analysis indicates that
the upregulation of miR-125a/b and miR-23a/b should produce repression of
connexin43 and connexin45 and the down regulation of miR-1, miR-30a/b/c and miR-
150 should do the opposite. These two opposing effects may cancel out each other.
5. Prolongation of ventricular APD is typical of heart failure to enable the improvement of
contraction strength, thereby supporting the weakened heart. However, APD
prolongation consequent to decreases in several repolarizing K
+
current (I
to1
, I
Ks
, and
I
K1
) in failing heart often results in occurrence of early afterdepolarizations (EADs)
(Beuckelmann et la., 1993; Tsuji et al., 2000). Our prediction failed to provide any
explanation at the miRNA level: None of the upregulated miRNAs may regulate the
genes encoding repolarizing K
+
channels. On the contrary, downregulation of miR-1
and miR-133 predict upregulation of KCNE1/minK and KCNQ1/KvLQT1,
respectively.
6. A majority of published studies showed a decrease in I
K1
in ventricular myocytes of
failing hearts (Beuckelmann et la., 1993; Rose et al., 2005). But whether KCNJ2/Kir2.1,
the major subunit underlying I
K1
, is downregulated remained controversial in previous
studies and the mechanisms remained obscured. One study noted decreased KCNJ2
mRNA expression but unaltered Kir2.1 protein level (Rose et al., 2005). With our
prediction, the upregulated miRNAs (miR-125, miR-214, miR-24, miR-29, and miR-195)
predict reduction of inward rectifier K
+
channel subunits including KCNJ2/Kir2.1,
KCNJ12/Kir2.2, KCNJ14/Kir2.4, and KCNK1/TWIK1, whereas the downregulated
miRNAs (miR-1 and miR-30a/b/c) predict increase in KCNJ2/Kir2.1.
In summary, our analysis of target genes for deregulated miRNAs in hypertrophy/CHF
may explain at least partly the enhanced cardiac automaticity (relief of HCN2 repression

and increased NCX1 expression) and reduced cardiac conduction (repression of Nav1.5).
But the data suggest that miRNAs are hardly involved in the abnormality of cardiac
repolarization in cardiac hypertrophy and heart failure since the genes for the repolarizing
K
+
channels were not predicted as targets for the upregulated miRNAs. The prediction of
NCX1 upregulation as a result of derepression from miRNAs may be of particular
importance aberrantly enhanced NXC1 activity has also been noticed in atrial fibrillation
occurring in CHF.
2.4 Application of our bioinformatics analysis to myocardial infarction (MI)
MI is manifested as cascades of electrical abnormalities and even lethal arrhythmias as a
result of deleterious alterations of gene expression outweighing adaptive changes
(Carmeliet, 1999). Ischemic myocardium demonstrates characteristic sequential alterations
in electrophysiology with an initial shortening of APD and QT interval during the early
phase (<15min) of acute ischemia and subsequent lengthening of APD/QT after a
prolonged ischemic period and chronic myocardial ischemia (Carmeliet, 1999; Nattel et al.,
2007). To exploit if miRNAs could be involved in the remodelling process, several original
studies have been published. We first identified upregulation of miR-1 in acute myocardial
infarction and the ischemic arrhythmias caused by this deregulation of miR-1 expression
(Yang et al., 2007). Subsequently, miRNA expression profiles in the setting of myocardial
ischemia/reperfusion injuries were reported by four groups (Dong et al., 2009; Luo et al.,
2010; Ren et al., 2009; Roy et al., 2009).
Based on these published data, we made an analysis to exclude that miRNAs that were
found deregulated by a study but not by others and that were found deregulated in rat heart
but was not expressed in human heart. In this way, we identified an array of miRNAs that
are likely deregulated in the setting of myocardial ischemia. The MI-upregulated miRNAs
include miR-1, miR-23, miR-29, miR-20, miR-30, miR-146b-5p, miR-193, miR-378, miR-181, miR-
491-3p, miR-106, miR-199b-5p, and let-7f; and the downregulated miRNAs include miR-320,
miR-185, miR-324-3p, and miR-214. Interesting to note is that some of the miRNAs
demonstrated the opposite directions of changes in their expression between ischemic
myocardium and hypertrophic hearts. For example, miR-1, let-7, miR-181b, miR-29a and miR-
30a/e are upregulated in ischemic myocardium, but downregulated in hypertrophy.
Similarly, miR-214, miR-320 and miR-351 are down-regulated in ischemic myocardium, but
up-regulated in hypertrophy. This fact further reinforces the notion that different
pathological conditions are associated with different expression profiles: miRNA signatures.
Our analysis yielded the following notions.
1. Six upregulated miRNAs (miR-1, miR-29, miR-20, miR-30, miR-193 and miR-181) were
predicted to target several Kir subunits (KCNJ2, KCNJ12, KCNJ, and KCNK1), but none
of the downregulated miRNAs can target these genes (Fig. 4). This is in line with the
previous finding that I
K1
is reduced and membrane is depolarized in ischemic
myocardium (Carmeliet, 1999; Nattel et al., 2007; Yang et al., 2007).
2. The cardiac slow delayed rectifier K
+
current (I
Ks
) is carried by co-assembly of an -
subunit KvLQT1 (encoded by KCNQ1) and a -subunit mink (encoded by KCNE1).
Loss-of-function mutation of either KCNQ1 or KCNE1 can cause long QT syndromes,
indicating the importance of I
Ks
in cardiac repolarization. In ischemic myocardium,
persistent decreases in minK with normalized KvLQT1 protein expression have been
observed which may underlie unusual delayed rectifier currents with very rapid

activation (Sanguinetti et al., 1996; Dun & Boyden, 2005), resembling currents produced
by the expression of KvLQT1 in the absence of minK. We have experimentally
established KCNE1 as a target for miR-1 repression [Luo et al., 2007], which was also
predicted in the present analysis. Moreover, no other miRNAs were predicted to target
KCNQ1. This finding is coincident with the observations on the diminishment of minK
alone without changes of KvLQT1 in ischemic myocardium.
3. It has been observed that cells in the surviving peri-infarct zone have discontinuous
propagation due to abnormal cell-to-cell coupling (Gardner et al., 1985; Peters, 1995; Spear
et al., 1983). This is largely due to decreased expression and redistribution of gap junction
protein connexins (Cxs). In this study, seven out of 12 upregulated miRNAs were
predicted to target Cxs including GJA1/Cx43, GJC1/Cx45, and GJA5/Cx40, but only one
downregulated miRNA miR-185 may regulate GJA5/Cx40. This result clearly points to
the role of miRNAs in damaging cardiac conduction in ischemic myocardium. Indeed,
repression of GJA1/Cx43 to slow conduction and induce arrhythmias in acute myocardial
infarction has been experimentally verified by our previous study (Yang et al., 2007).
4. In ischemic myocardium, fast or peak sodium current (I
Na
) density is reduced, which
may also account partly for the conduction slowing and the associated re-entrant
arrhythmias (Friedmanet al., 1975; Pu & Boyden, 1997; Spear et al., 1983). Our analysis
showed that let-7f and miR-378 may target SCN5A/Nav1.5 and upregulation of these
miRNAs is anticipated to cause reduction of I
Na
via downregulating SCN5A/Nav1.5 in
myocardial infarction. By comparison, none of the downregulated miRNAs may repress
SCN5A/Nav1.5 based on our target prediction.
5. Transient outward K
+
current (I
to1
) is reduced in myocardial ischemia and in rats, I
to1

decreases correlate most closely with downregulation of KCND2-encoded Kv4.2
subunits. miR-1 is predicted to repress KCND2/Kv4.2, and miR-29 may target KCHiP2
that is known to be critical in the formation of I
to1
.
6. L-type Ca
2+
current (I
Ca,L
) is diminished in border-zone cells of dogs. miR-30 family has
the potential to target CACNA1C/Cav1.2 and CACNB2/Cav2, and miR-124, miR-181,
miR-320 and miR-204 to target CACNB2. Upregulation of miR-30, miR-124 and miR-181
therefore would decrease CACNA1C/Cav1.2 and CACNB2/Cav2 expression, but
downregulation of miR-320 and miR-204 tends to increase the expression of these genes.
Considering the relative abundance of these miRNAs, it seems that the decreasing force
overweighs the increasing force with a balance towards a net inhibition of I
Ca,L
.
7. Na
+
/K
+
ATPase is a sarcolemmal ATP-dependent enzyme transporter that transports
three intracellular Na
+
ions to the extracellular compartment and moves two
extracellular K
+
ions into the cell to maintain the physiological Na
+
and K
+

concentration gradients for generating the rapid upstroke of the action potential but
also for driving a number of ion-exchange and transport processes crucial for normal
cellular function, ion homeostasis and the control of cell volume. It is electrogenic,
producing a small outward current I
P
. We noticed that the ischemia-induced
upregulation of miR-29 and miR181 expression might render inhibition of Na
+
/K
+

ATPase activity as they possibly target the ATP1B1 -subunit of the enzyme. This may
contribute to the electrical and contractile dysfunction in the ischemic/reperfused
myocardium due to the ischemia-induced inhibition of the Na
+
/K
+
ATPase and the
failure of intracellular Na
+
to recover completely on reperfusion [Fuller et al., 2003].
In a whole, it appears that the expression signature of miRNAs in the setting of myocardial
ischemia and the predicted gene targeting of these miRNAs coincide with the ionic
remodelling process under this pathological condition. The miRNAs seem to be involved in

all aspects of the abnormalities of cardiac excitability during ischemia, as manifested by the
slowing of cardiac conduction due to reduced I
Na
and Cx43, the depolarized membrane
potential to adversely affect cardiac conduction due to reduced I
K1
, the impaired excitation-
contraction coupling and contractile function due to reduced I
Ca,L
and Na
+
/K
+
ATPase, and
the delayed cardiac repolarization due to reduced I
Ks
and I
to1
.
3. Apoptosis-related genes as argets for miRNAs
It has been nearly 40 years since Kerr named the novel death process apoptosis, from the
Greek word meaning falling of the leaves, an active process that leads to cell death (Kerr et
al., 1972). The human body destroys ~60x10
9
cells/day through an apoptotic process in
response to various stresses such as physiological, pathogenic, or cytotoxic stimuli (Reed,
2002). Unlike necrosis, apoptosis is a complex endogenous gene-controlled event that requires
an exogenous signalstimulated or inhibited by a variety of regulatory factors, such as
formation of oxygen free radicals, ischemia, hypoxia, reduced intracellular K
+
concentration,
and generation of nitric oxide. Progressive cell loss due to apoptosis is a pathological hallmark
implicated in a wide spectrum of degenerative diseases such as heart disease, atherosclerotic
arteries and hypertensive vessels, Alzheimers disease, etc (Jaffe et al., 1997; Palojoki et a., 2001;
Sabbah et a., 1998). Apoptosis as an early and predominant form of cell death has been
detected in human acute myocardial infarcts and it was shown to increase in reperfused
myocardium. Apoptosis is also believed to account for the loss of cell mass in failing heart.
Evidence for the role of miRNAs in cardiomyocytes apoptosis has been rapidly accumulating.
My group documented the first of such evidence; the muscle-specific miRNAs miR-1 and miR-
133 produce opposing actions on cardiomyocyte apoptosis with the former being proapoptotic
while miR-133 being antiapoptotic (Xu et al., 2007). miR-21 is also an antiapoptotic miRNA. It
has been shown to produce beneficial effects against H
2
O
2
-induced injury on cardiac myocytes
and ischemia/reperfusion injury via antiapoptosis through its target Programmed Cell Death
4 (PDCD4). Based on our computational prediction, many other miRNAs, such as the miR-
17~92 cluster and its two paralogs miR-106a~363 and miR-106b~25 clusters, also have the
potential to regulate cardiomyocyte apoptosis by targeting the related genes in the signalling
pathways (unpublished observations).
Following similar procedures we used to predict ion channel genes as targets for miRNAs
described in section 2.1, we analyzed the genes known to be crucial for cell survival and
death for miRNA regulation.
3.1 Control of cardiomyocyte apoptosis by miRNAs under normal conditions
Our analyses allowed us to have an overall picture on how the cardiomyocyte homeostasis
may be maintained by miRNAs and to divide miRNAs roughly into two groups: pro-
apoptotic miRNAs and anti-apoptotic miRNAs, though there is no clear-cut distinction as each
miRNA may simultaneously target both survival and apoptotic genes. This property indicates
that cardiomyocyte survival and death is tightly controlled and delicately balanced. Any
changes of expression of miRNAs can shift the balance leading to alterations of cell fate.
1. Among the top 20 most abundant miRNAs in the heart, only miR-99 and miR-100 have
no predicted target genes relevant to apoptosis and others have 1 to 27 targets.
2. The let-7 family, miR-16, miR-20a/b, miR-125a/b, and miR-29a/b were predicted to
some major survival genes including BCL2, BCL2L2, AKT2, AKT3, STAT3, IGF0-1 and
MCL1. Thus, they are more likely to be pro-apoptotic miRNAs. Indeed, miR-29 has

been experimentally proven to be pro-apoptotic (Ye et al., 2010). And our unpublished
observations indicate a strong promotion of cardiomyocyte apoptosis by miR-20a/b.
The studies conducted in cancer cells support our notion that miR-16 induces apoptosis
(Cimmino et al., 2005; Tsang & Kwok, 2010), though its effects on cardiac cells have not
yet determined.
3. The miR-30 family, miR-24, miR-23a/b, miR-26a/b, miR-27a/b, miR-145, miR-92a/b,
and miR-199a/b may be anti-apoptotic miRNAs as they were predicted to target many
important cell death genes, such as CASP3 (encoding caspase 3), CASP7, BCL2L11,
BAK1, BAX, FOS, etc. Among these miRNAs, miR-199 and miR-24 have been shown to
produce cardioprotective effects against apoptosis (Qian et al., 2011). miR-145 is known
to mediate inhibition of proliferation and induction of apoptosis of cancer cells
(Ostenfeld et al., 2010; Sachdeva & Mo, 2010).
4. Several miRNAs were predicted to target both survival and apoptotic genes; these include
the muscle-specific miRNAs miR-1, miR-133, miR-21, miR-195. In theory, these miRNAs
are neutral without affecting ell death or can produce either pro-apoptotic or anti-
apoptotic effect depending on particular cellular context: expression of particular target
genes for a particular miRNA. Indeed, miR-1 and miR-133 do not affect cardiomyocyte
apoptosis under normal conditions, but when many survival and death genes are
increased in their expression in response to oxidative stress, miR-1 promotes
cardiomyocyte apoptosis by targeting heat shock protein 60 whereas miR-133 protects
against apoptosis by repressing caspase 9 (Xu et al., 2007). miR-21 has been commonly
believed to elicit cardioprotective effects in myocardial ischemia and
ischemia/reperfusion injuries (). But in tumor cells it has been reported to be pro-
apoptotic, anti-apoptotic or neutral. For example, knockdown of miR-21 in cultured
glioblastoma cells resulted in a significant drop in cell number. This reduction was
accompanied by increases in enzyme activity of caspases 3 and 7, as well as terminal
deoxyribonucleotidyl transferase-mediated dUTPdigoxigenin nick end-labelling
(TUNEL) staining (Chan et al., 2005; Corsten et al., 2007). In MCF-7 human breast cancer
cells, miR-21 elicits anti-apoptotic effects (Si et al., 2007; Zhu et al., 2007). However, in
neuroblastoma cells, miR-Lat was reported to protect against apoptotic cell death (Gupta
et al., 2006). This property of miR-21 in cancer cells is in line with our prediction.
5. The cardiac-specific miRNAs miR-208a/b and miR-499 do not seem to have significant
role in regulating apoptosis since they were predicted to target only a small number of
genes involved apoptosis signalling: CDKN1A and E2F6 whose expression levels are
low in heart. Moreover, the expression levels of these cardiac-specific miRNAs are also
in the low range.
3.2 Control of cardiomyocyte apoptosis by miRNAs in ischemic myocardium
Myocardial infarction (MI), a typical situation of metabolic stress, is presented as cascades of
cellular abnormalities as a result of deleterious alterations of gene expression outweighing
adaptive changes. MI can cause severe cardiac injuries and the consequences are contraction
failure, electrical abnormalities and even lethal arrhythmias, and eventual death of the cell.
Apoptosis is an important mechanism for the cell death occurring in ischemic myocardium.
Previous work on miRNAs and apoptosis has been mostly limited to the context of cancer,
while studies on apoptosis regulation by miRNAs in non-cancer cells have been sparse. The
first evidence for the role of miRNAs in cardiomyocyte apoptosis was obtained in 2007 from
my laboratory demonstrating the proapoptotic effect of miR-1 and anti-apoptotic effect of

miR-133 in response to oxidative stress (Xu et al., 2007), with miR-1 causing proapoptotic
effects confirmed by other groups (Yu et al., 2008; Tang et al., 2010). Subsequent studies in
2009 and 2010 revealed the involvement of other miRNAs such as miR-21, miR-24, miR-29,
miR-199a, and miR-320 in ischemic myocardial injury (Cheng et al., 2010; Qian et al., 2011;
Rane et al., 2009; Ren et al., 2009; Ye et al., 2010; Yin et al., 2008).
Extracting of the overlapping results from different laboratories and filtering with the
cardiac expression profile verified by real-time RT-PCR in human hearts allowed us to
identify an array of miRNAs that are likely deregulated in the setting of myocardial
ischemia. The upregulated miRNAs include miR-1, miR-23, miR-29, miR-20, miR-30, miR-
146b-5p, miR-193, miR-378, miR-181, miR-491-3p, miR-106, miR-199b-5p, and let-7f; the
downregulated miRNAs include miR-320, miR-185, miR-324-3p, and miR-214. We then
applied our procedures to these miRNAs and our analyses yielded the following notions.
1. Among the upregulated miRNAs, only miR-99 and miR-100 have no predicted target
genes relevant to apoptosis and others have 1 to 27 targets related to cell survival and
death. Notably, a majority of these miRNAs are predicted to be pro-apoptotic: let-7f,
miR-1, miR-20, miR-29, miR-106, miR-181, miR-193, miR-378 and miR-491-3p, leaving
the other four (miR-23, miR-30, miR-146b-5p and miR-199b-5p) being anti-apoptotic.
2. Among the downregulated miRNAs, except for miR-185 which is expressed with
extremely low abundance, miR-214, miR-320 and miR-324 are supposed to be neutral as
they were predicted to target both survival (AKT3, STAT3 and MCL1) and apoptotic
(CASP3, BAX, CDK6, etc) genes. Their downregulation therefore may not cause
significant impact on cell death in MI. However, it has been reported that
overexpression of miR-320 in cultured adult rat cardiomyocytes enhanced apoptotic cell
death, whereas knockdown produced cytoprotective effect against apoptosis, on
simulated ischemia/reperfusion injuries, through targeting HSP20 (Ren et al., 2009),
which is not within the list of our present theoretical prediction.
3. Taken together, it appears that the pro-apoptotic force is enhanced more than the anti-
apoptptoic force, being in agreenment with the fact that apoptosis is increased in the
setting of MI.
4. Fibrosis-related genes as targets for miRNAs
In tissues composed of post-mitotic cells, like heart, new cells cannot be regenerated;
instead, fibroblasts proliferate to fill the gaps created due to removal of dead cells. In the
normal heart, two thirds of the cell population is composed of nonmuscle cells, the majority
of which are fibroblasts (Maisch, 1995; Manabe et al., 2002). Cardiac fibroblasts, along with
cardiomyocytes, play an essential role in the progression of cardiac remodelling. Damaging
insults evoke multiple signalling pathways that lead to coordinate and sequential gene
regulation; the initial events lead to the activation of cardiac fibroblasts. Cardiac fibrosis is
the result of both an increase in fibroblast proliferation and extracellular matrix (ECM)
deposition. Cardiac myocytes are normally surrounded by a fine network of collagen fibres.
Myocardial fibrosis is an established morphological feature of the structural myocardial
remodelling that is a characteristic of all forms of cardiac pathology (Berk et al., 2007; Khan &
Sheppard, 2006). A growing body of evidence indicates that, along with cardiomyocytes
hypertrophy, diffuse interstitial fibrosis is a key pathologic feature of myocardial remodelling
in a number of cardiac diseases of different (e.g. ischemic, hypertensive, valvular, genetic, and
metabolic) origin. Acute myocardial infarction due to coronary artery occlusion represents a

major cause of morbidity and mortality in humans (Fox et al., 2007). The loss of blood flow to
the left ventricular free wall of the heart after MI results in death of cardiomyocytes and
impaired cardiac contractility. Scar formation at the site of the infarct and interstitial fibrosis of
adjacent myocardium prevent myocardial repair, diminish coronary reserve and contribute to
loss of pump function, and predisposes individuals to ventricular dysfunction and
arrhythmias, which, in turn, confer an increased risk of adverse cardiovascular events
(Swynghedauw, 1999). Elucidation of the precise mechanisms responsible for the actions of
these factors could forge new frontiers in both risk identification and prevention of fibrosis-
derived clinical complications in patients with cardiac disease.
A subset of miRNAs is enriched in cardiac fibroblasts compared to cardiomyocytes. A
number of studies have demonstrated the involvement of miRNAs in regulating myocardial
fibrosis in the settings of myocardial ischemia or mechanical overload. In this conceptual
framework, the investigation of miRNAs might offer a new opportunity to advance our
knowledge of the pathogenesis of fibrosis. Characterization of individual miRNAs or
miRNA expression profiles that are specifically associated with myocardial fibrosis might
allow us to develop diagnostic tools and innovative therapies for fibrogenic cardiac diseases.
The identification of miRNAs as potential regulators of myocardial fibrosis has clinical
implications; the search for a miRNA expression pattern specific to fibrosis might provide a
novel diagnostic approach. Yet, the molecular mechanisms that lead to a fibrogenic cardiac
phenotype are still being elucidated.
4.1 Control of cardiac fibrogenesis by miRNAs under normal conditions
Our analysis revealed that 19 of the 20 most abundant miRNAs in the heart have the
potential to repress multiple genes known to be involved in fibrogenesis including various
types of collagens (COL), CTGF (connective tissue growth factor), FBN1/2/3
(fibrillin1/2/3), ASPN (asporin), MMP2 (matrix metallopeptidase 2), FN1 (fibronectin 1),
and various types of TRP channels (transient receptor potential). miR-126 is the only one
among the 20 most abundant miRNAs that was not predicted to regulate any fibrosis-
relevant genes. The cardiac-specific miRNAs miR-208a and miR-208b seem to have minimal
effects on fibrosis sine they have only two target genes OMG (oligodendrocyte myelin
glycoprotein) and TTN (titin). Another cardia-specific miRNA miR-499 is likely an anti-
fibrotic miRNA as it was predicted to target 12 profibrotic factors including collagens,
LAMA1 (laminin 1), FBN2, FN1, OMG, SLN (sarcolipin), TTN, etc. It should be noted that
all these target genes encode profibrotic proteins. Our data therefore indicate that the heart
is evolved with a super-strong epigenetic program to prevent fibrogenesis or to suppress
fibrosis under normal conditions.
Experimentally, several miRNAs including miR-29, miR-30, miR-133 and miR-590 were all
found to produce anti-fibrotic effects (Duisters et al., 2009; Shan et al., 2009; van Rooij et al.,
2008), whereas evidence exists for miR-208 (van Rooij et al., 2007) and miR-21 as pro-fibrotic
miRNAs (Roy et al., 2009; Thum et al., 2008; van Rooij et al., 2008;). The former can be
explained based on our prediction that miR-29, miR-30, miR-133 and miR-590 all have the
potential to target profibrotic genes. The latter, however, seems not quite straightforward.
Surprisingly, a murine genetic miR-21 knockout model failed to show an antifibrotic
phenotype after cardiac stress suggesting differences in pharmacological and genetic miR-21
knockdown (Patrick et al., 2010). Indeed, the various miR-21 inhibitor chemistries have
different effects on cardiac fibrosis (Thum et al., 2011). It is likely that other genes not included
in our analysis may be the targets for miR-208 and miR-21 to produce pro-fibrotic actions.

4.2 Control of atrial fibrogenesis by miRNAs during atrial fibrillation
Atrial fibrillation (AF) is the most commonly encountered clinical arrhythmia that causes
tremendous health problems by increasing the risk of stroke and exacerbating heart failure.
It is characterized by a process termed atrial structural remodelling with increased atrial
fibrosis. Indeed, atrial fibrosis has been strongly associated with the presence of heart
diseases/arrhythmias, including congestive heart failure (CHF) and AF (Pellman et al., 2010;
Tan & Zimetbaum, 2010).
To determine if miRNAs are involved in atrial structural remodelling, we first conducted
expression profiling to identify deregulated miRNAs in the atrial tissues of a canine model
of tachypacing-induce chronic AF, using miRNA microarray analysis comparing the
differential expressions of miRNAs between control and AF dogs. Four miRNAs miR-223,
miR-328, miR-664 and miR-517 were found increased by >2 folds, and six were decreased by
at least 50% including miR-101, miR-133, miR-145, miR-320, miR-373 and miR-499. Real-time
quantitative RT-PCR (qRT-PCR) analysis confirmed the significant upregulation of miR-223,
miR-328 and miR-664 (miR-517 was undetectable), and the significant downregulation of
miR-101, miR-320, and miR-499. Intriguingly, none of these deregulated miRNAs are within
the list of top 20 most abundant miRNAs. But miR-223 and miR-328 are among the cardiac-
enriched miRNAs. This notion would suggest that altered miRNA expression in this AF
model tends to favour fibrogenesis; however, miRNAs are definitely not the major
determinant for atrial structural remodelling associated with fibrosis.
In a recent study reported by Chens group (Xiao et al., 2011), it was found that miRNA
expression undergoes tremendous alterations in atrial tissues from AF patients with mitral
stenosis. Intriguingly, out of 20 most abundant miRNAs in the heart, only let-7b/i and miR-
30d were found significantly upregulated but 9 of 20 including miR-29, miR-133, miR-24,
miR-26, miR-126, miR-125, miR-99, miR-20, and miR-23 were downregulated. Based on our
computational prediction, these changes are expected to result in reduction of the anti-
fibrotic force to promote atrial fibrogenesis.
5. Conclusion
The theoretical analyses in conjunction with experimental demonstration of miRNA
expression profiles under various conditions performed presented here allowed us to
establish a matrix of miRNAs that are expressed in cardiac cells and have the potential to
regulate the genes encoding cardiac ion channels and transporters, proteins responsible for
cell survival and death, and proteins involved in fibrogenesis in heart. These miRNAs likely
play an important role in controlling cardiac excitability, cardiomyocyte homeostasis and
cardiac fibrosis of the heart. In other words, the genes determining these processes may
normally be under the post-transcriptional regulation of a group of miRNAs. Indeed, some
of the predicted targets have already been demonstrated experimentally. Also we were able
to link a particular remodeling process in hypertrophy/heart failure, myocardial ischemia,
or atrial fibrillation to the corresponding deregulated miRNAs under that pathological
condition; the changes of miRNAs appear to have anti-correlation with the changes of many
of the genes responsible for cardiac electrophysiology, cardiomyocyte apoptosis and cardiac
fibrosis under these situations. The present study should aid us to pinpoint the individual
miRNAs that can most likely take part in the electrical and structural remodelling processes
through targeting particular genes.
It should be noted, however, that the present computational study is in no way to replace
experimental approaches for understanding the role of miRNAs in regulating expression of

genes; rather it merely presents a prediction of the odds of miRNA:mRNA interactions
under normal situation and in the context of electrical/ionic remodeling under the selected
circumstances of the heart. This theoretical analysis like all other computational studies
needs to be eventually verified with the bench-top work and should not be considered
original results. Nonetheless, with sparse experimental data published to date and the
anticipated difficulties to acquire complete experimental data using the currently available
techniques, the analytical procedures described here can well serve as first-hand
information, providing a framework and guideline for future experimental studies.
The second limitation of the study is the possibility of underestimating the number of
miRNAs that could regulate ion channels, apoptosis and fibrosis due to the stringent
criterion for inclusion of miRNAs with positive prediction of targets by at least four out of
seven algorithms; in the past, we had been able to experimentally verified nearly all the
target genes predicted by only one algorithm miRanda for our pre-experiment analysis.
However, the fact that our prediction includes all 20 most abundant miRNAs and other
highly expressed miRNAs in the myocardium suggests that this limitation might not have
significant negative impact on the accuracy of our analysis and inclusion of more miRNAs
by more permissive criteria does not guarantee their physiological function if they are
scarcely expressed in the heart. Yet it should be noted that the miRNA expression profiles
were obtained from myocardium that also includes fibroblasts and caution needs to be
taken when interpreting the expression data.
Another important notion is that despite that our prediction of miRNA targeting coincides
with the changes of expression of relevant genes under the pathological conditions, it does
not imply that miRNAs are necessarily the important or even the only determinant of the
electrical remodeling processes. Our data to the most indicate the potential contribution of
miRNAs to such conditions; other molecules like transcription factors must also be involved
in the regulation of expression of ion channel genes under these conditions.
Finally, it is also difficult to predict the net outcome when two miRNAs target a same gene
but alter in their expression in the opposite directions. Yet, with deepened and broadened
understanding of miRNA targeting and action, these possible limitations should eventually
be worked out.
6. Acknowledgment
The work presented was supported by the Canadian Institute of Health Research (CIHR).
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25
Genome-Wide Identification of Estrogen
Receptor Alpha Regulated miRNAs Using
Transcription Factor Binding Data
Jianzhen Xu
1
, Xi Zhou
2
and Chi-Wai Wong
3
1
College of Bioengineering, Henan University of Technology, Zhengzhou,
2
Guangzhou Institute of Biomedicine and Health, Chinese Academy of ScienceGuangzhou,
3
NeuMed Pharmaceuticals Limited, Hong Kong,
China
1. Introduction
MicroRNAs (miRNAs) are one class of endogenous non-coding RNA which can repress
protein translation or cause target mRNA degradation(Bartel 2004). Currently 15,172 entries,
including 1,048 human miRNAs, are recorded in a major miRNAs database miRbase
(Release 16: Sept 2010) (Kozomara and Griffiths-Jones 2011). MiRNAs reside reside in
protein-coding, intronic and intergenic regions throughout the genome. MiRNAs are mainly
transcribed into long primary miRNAs (pri-miRNAs) by RNA polymerase II(Lee et al. 2004).
Since mammalian miRNA genes are often clustered along the genome, the pri-miRNA can
contain one single miRNA gene or multiple clustered miRNA genes. In the nucleus, pri-
miRNAs, which are both capped and polyadenylated, are processed by RNase III enzymes
Drosha into about 70-nucleotide hairpins called pre-miRNAs(Lee et al. 2002). The
transporter protein exportin-5 then exports pre-miRNAs to the cytoplasm, where they are
cleaved by another RNase III Dicer to generate mature miRNA duplexes. One strand of
miRNA duplex preferentially enters into miRNA-induced silencing complexes (miRISCs)
and guides the complex to recognize its target genes. Previous studies indicated that this
target inhibition of miRNAs mainly function via imperfect base pairing with the targeting
sequences on the 3 untranslated region(3'UTR) and the first 28 bases of a particular mature
miRNA sequence referred to the seed region. MiRNAs play essential regulatory roles in
diverse biological processes. For example, we recently found that miRNA-153, the
expression level of which is significantly repressed in glioblastoma (GBM), could inhibit cell
proliferation and induce apoptosis via targeting B-cell lymphoma 2 (Bcl-2), myeloid cell
leukemia sequence 1 (Mcl-1) and insulin receptor substrate-2 (Irs-2) in glioblastoma cell
lines(Xu, Liao, and Wong 2010; Xu et al. 2011).
In the past few years, computational approaches have played an important role in miRNA
studies, for example, dozens of prediction tools used for miRNA gene finding and miRNA
target prediction were developed. These tools have greatly facilitated experimental
discovery. However, knowledge about the regulation of these essential regulators is at its
early stage(Schanen and Li 2010; Li et al. 2010). Transcriptional regulations mediated by
specific transcriptional factors (TFs) have only been intensively studied on a small number
of miRNAs(Lee et al. 2004; Houbaviy et al. 2005). Importantly, certain oncogenic miRNAs


560
and tumour suppressor miRNAs are inappropriately expressed in cancers. However, our
understanding as to the TFs or chromatin modifications responsible for governing the
expression levels of these essential miRNAs remains limited.
At the transcriptional level, gene expression is governed by interactions among TFs and cis-
elements such as promoters and enhancers. Chromatin immunoprecipitation (ChIP)
experiment discovers specific protein-DNA interactions in a given cell type and is regarded
as a major tool for investigating interactions between TFs and their binding sites. Based on
the pairing of ChIP with DNA microarray and high-throughput sequencing technologies
(ChIP-chip and ChIP-seq), genome-wide maps of TF binding sites can now be readily
produced. Many groups have used ChIP-chip and ChIP-seq assays to globally study direct
targets of TFs and provided significant insights into gene regulation networks (Farnham
2009). Together with mRNA-based expression microarrays, vast amounts of data are
publicly available for analysis by bioinformatics. Indeed, networks of gene expression (or
systems biology) are gaining popularity to help uncover the physiology regulation
underneath and interpret the biological meaning behind these networks.
In principle, one can use the genome-wide binding map of a specific TF (or a chromatic
modifying factor) to search for its putative target miRNAs, i.e. locate putative binding sites
inside miRNA regulatory regions (such as promoter or enhancer) according to genomic
coordinates. In the following section, we present a procedure which uses published ChIP-
chip data to predict candidate miRNAs regulated by a specific TF. Specifically, based on one
genome-wide estrogen receptor (ER) binding map, we found 59 miRNA regulatory regions
in which there is at least one ER binding site. Several putative ER-regulated oncogenic and
tumour suppressor miRNAs were further confirmed in a breast cancer cell model.
2. Methods
2.1 Prediction of ER-regulated miRNAs
Accessing and analyzing the genomic sequence and functional annotations were based on
the UCSC Genome Browser(Rhead et al. 2010) and Galaxy platform(Goecks, Nekrutenko,
and Taylor 2010). UCSC Genome Browser is a web tool for convenient displaying and
accessing the genome sequences, together with rich annotation tracks. Galaxy platform is an
interactive system that combines existing multiple genome resources via a simple web
portal. Users can manipulate remote resources and perform flexible operations such as
intersections, unions, and subtractions. Currently, 718 miRNAs have been annotated in
human genome (hg18). Their regulatory regions (50 Kb upstream from the pre-miRNAs)
were collected from UCSC Genome Browser. The original ChIP-chip data were produced by
Carroll et al. (Carroll et al. 2006). Totally 3,665 estrogens receptor binding regions, which
were considered with high confidence, were used in this study. Since the original
coordinates were annotated in hg17, a web-based liftOver utility with default settings was
used to convert the genomic coordinates to the hg18 version of the human genome
(http://genome.ucsc.edu/cgi-bin/hgLiftOver). Overlapping regions were searched in
Galaxy platform.
2.2 Motif analysis
The ER binding regions which overlapped with miRNA regulatory sequences were
download from UCSC Genome Browser and further analyzed by TOUCAN 2, a widely used
regulatory sequence analysis suite (Aerts et al. 2005). It screened the input sequences against
a precompiled library of motifs to find the statistically over-represented motifs. TRANSFAC
Genome-Wide Identification of Estrogen Receptor Alpha
Regulated miRNAs Using Transcription Factor Binding Data

561
is a database storing eukaryotic transcription factors and the transcription regulating DNA
sequence elements(Matys et al. 2006). Position weight matrixes (PWMs) were obtained from
the TRANSFAC 7.0 database. The Eukaryotic Promoter Database (EPD) collected annotated
eukaryotic RNA polymerase II promoters sequences around the experimentally determined
transcription start site(Schmid et al. 2006). The human promoter sequence (-499,100 around
TSS) from EPD were used as background sequences. The 0th order of the Markov model
with prior 0.1 was chosen to compute both the background sequences and the actual
sequence frequencies. The p-value and significance value indicate the probability that the
observed over-representation of the motif is achieved by random selection for a single or
multiple TFs, respectively.
2.3 Cells culture, cell counting and qRT-PCR
MCF-7 cell line was from American Type Culture Collection (Manassas, VA). Cells were
grown with phenol red-free D-MEM supplemented with 0.5% charcoal stripped FBS for 3
Days. The estrogen-deprived MCF-7 cells were treated with 10 nM 17-estradiol (E2, Sigma-
Aldrich Co.) or DMSO as a control. At the indicated time points, cells were rinsed with PBS
and counted manually under the microscope. Total RNA was collected and extracted with
Trizol reagent (Invitrogen). Reverse transcription of mature miRNAs and quantitative real-
time PCR analysis were performed as previously reported(Xu, Liao, and Wong 2010).
Primers specific for the indicated miRNAs were available upon request.
3. Results
3.1 Identifying putative miRNAs regulated by ERs
Estrogen receptor alpha (ER) and beta (ER) are members of the nuclear receptors super-
family which are ligand-regulated transcription factors. Estrogen (17-estradiol, E2) is a
potent ligand for both ERs. ERs either directly interact with cis-regulatory elements of target
genes by binding to estrogen-response elements (EREs) or indirectly tether to transcription
factors such as AP1 and SP1 (Ali and Coombes 2002). By transcriptional control of a large
number of target genes, ERs regulate a wide variety of cellular processes including
development and differentiation (Deroo and Korach 2006). In particular, ER is thought to
be involved in the progression of breast cancer. Depending on the status of ER expression,
breast cancer is classified into ER+ and ER- subtypes. Differential anti-hormone
treatments are prescribed in conjunction with anti-cancer drugs to manage these breast
cancer subtypes. Therefore, understanding how ER affects the expressions of oncogenic and
tumour suppressor miRNAs may assist better development of anti-cancer therapy.
Carroll et al. previously used ChIP-chip technology to analyze ER binding regions genome-
wide. They found that the majority of ER binding regions are located outside of the classical
promoter-proximal regions, suggesting distal regulation by ER (Carroll et al. 2006). To
account for the possible bias when only focusing on promoter-proximal regions, we
regarded 50 Kb upstream of all known pre-miRNAs as possible regulatory regions. Besides,
setting a wider candidate region should be beneficial at this exploring step. Totally 59
miRNA regulatory regions were found that overlapped with 65 Carrolls ER binding regions
(Table 1). As shown in Figure 1, there are three representative patterns of ER binding
regions relative to specific miRNAs. For example, the promoter of hsa-miR-342 contains
both promoter-proximal and distal ER binding regions, whereas the promoter of hsa-miR-21
is characterized by two proximal ER binding regions. In contrast, there is only one distal ER
binding region within the miR-143~145 cluster upstream regulatory region.


562
chromosome chrStart chrEnd miRNA regulatory regions strand
chr1 149734895 149784895 hsa-mir-554_up_50000_chr1_149734896_f +
chr1 154656845 154706845 hsa-mir-9-1_up_50000_chr1_154656846_r -
chr1 153381591 153431591 hsa-mir-92b_up_50000_chr1_153381592_f +
chr10 134911115 134961115 hsa-mir-202_up_50000_chr10_134911116_r -
chr12 96431720 96481720 hsa-mir-135a-2_up_50000_chr12_96431721_f +
chr12 63252555 63302555 hsa-mir-548c_up_50000_chr12_63252556_f +
chr15 61950271 62000271 hsa-mir-422a_up_50000_chr15_61950272_r -
chr17 26876542 26926542 hsa-mir-365-2_up_50000_chr17_26876543_f +
chr19 10789172 10839172 hsa-mir-199a-1_up_50000_chr19_10789173_r -
chr5 41461490 41511490 hsa-mir-1274a_up_50000_chr5_41461491_f +

563
chr7 101833307 101883307 hsa-mir-548o_up_50000_chr7_101833308_r -
chr1 153381591 153431591 hsa-mir-92b_up_50000_chr1_153381592_f +
chr12 63252555 63302555 hsa-mir-548c_up_50000_chr12_63252556_f +
chr15 61950271 62000271 hsa-mir-422a_up_50000_chr15_61950272_r -
chr19 10789172 10839172 hsa-mir-199a-1_up_50000_chr19_10789173_r -


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chr5 41461490 41511490 hsa-mir-1274a_up_50000_chr5_41461491_f +
chr7 101833307 101883307 hsa-mir-548o_up_50000_chr7_101833308_r -
Table 1. miRNA regulatory regions overlapped with ER binding regions. Each miRNA
regulatory region was annotated with chromosome, start and end position and the strand it
resides.

Fig. 1. ER binding sites relative to specific miRNAs. The blue boxes represent ER binding
regions and the black blocks represent upstream 50 Kbp of miRNAs. The pre-miRNAs, miR-
342, miR-21, miR-143~145 were represented by red colour. To note, there are two ER
binding regions within miR-342 and miR-21 regulatory regions. Correspondingly, miR-143
and miR-145 share the same ER binding region.

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We then used TOUCAN 2 to see whether there are TFs binding sites over-represented in
these miRNA-related ER-binding regions. The top 10 significant binding motifs are listed in
Table 2. Not surprisingly, we identified the consensus ERE (AGGTCANNNTGACC) as the
most common TF binding motif presented in these miRNA regulatory regions bound by ER.
In addition, we also observed enrichments of activator protein 1(AP-1) and forkhead (FKH)
motifs among the miRNA-related ER-binding regions. The AP-1 family consists of proteins
belonging to the JUN, FOS and ATF subfamilies. These subunits can hetero-dimerize and
bind to their DNA target genes. AP-1 complex modulates a variety of cellular processes in
response to environmental stimuli. Specially, AP-1 complex is an important regulator in
tumour development since its target genes are involved in oncogenic transformation,
tumour suppression, invasive growth and angiogenesis(Wagner 2001; Jochum, Passegue,
and Wagner 2001). FKH proteins are a super-family of transcription factors that participate
in regulating the expression of genes involved in cell growth, proliferation and
differentiation. Many FKH proteins are important to embryonic development, glucose
homeostasis, tumourigenesis and even vocal learning (Hannenhalli and Kaestner 2009). In
previous analysis of mRNA targets, these two binding motifs were also shown to enriched
in ER binding regions, suggesting their role in ERregulated mRNAs transcription (Carroll
et al. 2006). Our findings further implied that AP-1 and forkhead family members are
cooperating transcription factors to regulate ER responsive miRNAs in combinatorial
fashions.
In our result, p53 motif is the fourth most significant enriched binding sites in ER binding
regions. p53 is an essential tumour suppressor because mutations or aberrations in the
expression of p53 gene were frequently observed in a variety of cancer cell lines and clinical
tumour samples(Nigro et al. 1989). Liu et al. also found that ER can bind directly to p53
and repress its target genes (Liu et al. 2006). This important finding has profound
translational implications because the same group of investigators recently demonstrated
that (1) Ionizing radiation disrupts the ER-p53 interaction in breast tumours, functionally
leads to p53 restoration in breast tumours subjected to radiation therapy and elucidates a
novel mechanism underlying the anti-tumour effect of radiation therapy(Liu et al. 2009); (2)
The presence of wild-type p53 is an important determinant for responsiveness to anti-
estrogen therapy since anti-estrogens could reactivate p53 by disrupting the ERp53
interaction and subsequently p53 activates many tumour suppressor genes(Konduri et al.
2010). Similar with the situation for mRNA target regulation, we therefore hypothesize that
ERp53 interaction may also involve in modulating the transcription of oncogenic
miRNAs and tumour suppressor miRNAs although the exact mechanism needs further
analysis. Except for co-regulator of ER, there is also possible interaction between the
enriched TFs. For example, cross-talk between glucocorticoid receptor (GR) and AP-1 has
been well established (Herrlich 2001). In our result, both GR and AP-1 are also enriched in
the binding region, whether such interactions are involved in miRNAs target regulation
warrants further investigation.
In the original analysis of binding sites in mRNA promoters, Carroll et al. found there is a
strong correlation among ER, Forkhead, Oct, Ap-1 and C/EBP(Carroll et al. 2006). In our
analysis of miRNA promoter regions, we did not find significant enrichment for Oct and
C/EBP. But interestingly, several novel TF binding sites (v-Maf, Meis-1, p53, GR-, ROR1,
Hand1) are over-represented in miRNA-related ER-binding regions. This observation
perhaps reflect the similar (in the case of common TFs, i.e. ER, FHK and AP-1) and distinct
modes of ER modulation in miRNA and mRNA gene regulation.


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TRANSFAC Motif N P-value Sig value TFs
M00191 33 1.76E-13 10.338 ER-
M00035 21 1.52E-7 4.401 v-Maf
M00419 28 1.22E-6 3.498 Meis-1
M00272 21 2.32E-5 2.217 P53
M00192 34 4.13E-5 1.967 GR-
M00199 28 5.54E-5 1.84 AP-1
M00156 16 7.24E-5 1.723 ROR1
M00291 16 8.48E-5 1.655 FOXC1(Forkhead box protein C1)
M00222 22 1.86E-4 1.313 E47(Hand1)
M00269 24 2.14E-4 1.254 Xenopus fork head domain factor 3
Table 2. Top 10 enriched motifs in the miRNA-related ER-binding sites. N: number of times
TF site appears in the input sequences. Note that TF binding site might appear more than
once in one sequence. P-value: probability to find even more occurrences than N in the input
sequences. Sig value: a significance coefficient used to select the most overrepresented
patterns among the distinct motifs. When analyzing only one TF site, a P-value smaller than
0.05 could be considered as being over-represented. In case of multiple TF sites, sig-value is
used to select the significant result. Generally, positive sig values mean significant.
3.2 Confirmation of ER-regulated miRNA in a breast cancer cell model
MCF-7 is a well established ER+ cell line that reflects hormone-dependent breast cancer;
namely, E2 increases MCF-7 cell proliferation. In our hand, the cell number significantly
increased after four days of treatment with 10 nM E2 compared to DMSO control while a
late phase increase in cell number was observed starting on day 9 (Figure 2a). Among the
predicted E2-regulated miRNAs, we randomly selected 8 miRNAs and used qRT-PCR to
detect the time-dependent changes in their expression levels during cell proliferation.
Compared to DMSO control, miR-342, miR-21, miR-422a, miR-124, and miR-181c were
generally found to be up-regulated by E2 treatment; whereas miR-143, miR-145, and miR-
483 were down-regulated (Figure 2b and 2c), suggesting that they are under the respective
influences of positive and negative EREs. Intriguingly, the down-regulated miRNAs exhibit
wave patterns of expression, i.e., significantly suppressed on day 4 with differential levels of
restoration on day 7 followed by another round of suppression and partial rebound. Other
than miR-124 which displays a wave pattern of induction, the rest of the E2-induced
miRNAs show a gradual pattern of induction. The determinants and regulatory networks
that dictate these patterns of expression await comprehensive investigations.
Of those up-regulated miRNAs, miR-342 was induced to the highest extent by E2. MiR-342
is encoded in an intron of the gene EVL and commonly suppressed in human colorectal
cancer (Grady et al. 2008). Over-expression of miR-342 in the colorectal cancer cell line HT-
29 induced apoptosis, pointing towards a pro-apoptotic tumour suppressor function (Grady
et al. 2008). On the other hand, miR-342 expression level in breast tumours is more
complicated with highest level in ER and HER2/neu-positive luminal B tumours but lowest
level in ER, PR and HER2/neu triple-negative tumours (Lowery et al. 2009). Adding to the
uncertainty regarding its role, miR-342 is down-regulated in tamoxifen-resistant MCF-7 cells
(Miller et al. 2008). Consistent with these findings, Cittelly et al. compared miRNA

567
expression profiles between MCF-7/pcDNA (tamoxifen-sensitive) and MCF-7/HER216
(tamoxifen-resistant) cells when both cell lines were treated for 24 hr with 100 pM 17--
estradiol (E2) and 1M 4-hydroxytamoxifen (TAM). They found that miR-342 was the most
dramatically down-regulated miRNA in the tamoxifen resistant MCF-7/HER216 cells.
They further proved that other tamoxifen resistant cell lines such as TAMR1 and LCC2, all
exhibited dramatically suppressed levels of miR-342 whereas another tamoxifen sensitive
MCF-7/HER2 cell lines also expressed high levels of miR-342, indicating that loss of miR-
342 was a common feature of tamoxifen resistance(Cittelly et al. 2010).

Fig. 2. ER binding sites relative to specific miRNAs. (a) The effects of E2 (10 nM) on MCF-7
cell number over the course of 12 days are shown. (b-c) E2 induces expression of miR-342,
miR-21, miR-422a, miR-124 and miR-181c; whereas, decreases expression of miR-143, miR-
145 and miR-483 in MCF-7 cells. Cells were treated with E2 (10 nM) for indicated time and
miRNAs were subjected to qRTPCR analysis.
The expression level of miR-21 was previously found to be significantly changed in various
cancers; especially, it is higher in ER+ than ER breast tumour (Mattie et al. 2006; Volinia
et al. 2006). However, inconsistent results were reported regarding the effect of E2 on miR-
21 expression in MCF-7. Wickramasinghe et al. reported that E2 inhibited miR-21 expression
after 6 hr (Wickramasinghe et al. 2009). In contrast, another group found that miR-21 was
induced after a 4 hr E2 treatment (Bhat-Nakshatri et al. 2009). In our investigation, we found
that initially miR-21 was repressed after 4 days on E2. However, miR-21 was up-regulated


568
by E2 upon long term treatments. MiR-21 is thought to be an oncogenic miRNA (oncomiR)
and several confirmed endogenous targets such as PDCD-4 and PTEN are important tumor
suppressers (Asangani et al. 2008; Folini et al. 2010; Meng et al. 2007). Consistent with its
proposed role as an oncomiR, our results showed that miR-21 expression progressively
increased from day 7 to day 12 in parallel with the late phase increase in cell number.

Fig. 3. ER,AP-1 and p53 binding motifs relative to miR-342,miR-21 and miR-143~145. The
red, blue and pink boxes represent ER, AP-1 and p53 binding sites respectively. ER_2920
and ER2921 located in miR-342 upstream region; ER_3188 and ER_3189 located in miR-21
regulatory region; ER_1259 located in miR-21 upstream region (please referring to Figure 1).
MiR-143 and miR-145 are clustered miRNAs with their expression levels co-ordinately
down-regulated in multiple forms of cancer (Akao et al. 2007; Michael et al. 2003; Sevignani
et al. 2007; Wang et al. 2008). They can function as important tumour suppressers by
targeting multiple key genes in apoptosis, proliferation, and metastasis signalling pathways
(Chen et al. 2009; Chiyomaru et al. 2010; Sevignani et al. 2007; Zaman et al. 2010). However,
whether miR-143 and miR-145 are regulated by E2 in MCF-7 breast cell is unclear. In this
study, we found that both were repressed by E2 in a long term treatment. Importantly, we
also observed that ectopic expression of miR-145 repressed MCF-7 cell proliferation (data
not shown). These observations are consistent with previous studies in other cancers,
indicating that miR-145 is repressed in cancers compared to the normal control.
Analyzing the miR-342, miR-21 and miR-143~145 regulatory regions, we found AP-1
binding motifs in all of them, supporting the role of AP-1 as a basal activator (Figure 3)
(Wagner 2001). In addition, there are both ER and p53 motifs in the miR-342 upstream
region, therefore miR-342 may be a dual target of these two TFs and the expression of miR-
342 perhaps depends on both the integrity of estrogen signalling pathway but also the status
of p53. Estrogen-response elements were detected in both miR-342 and miR-143 promoter
regions, indicating direct estrogen receptor binding. However, ERE is not present in miR-21
upstream regulatory region and it is possible that transcriptional activation of miR-21 may
be mediated via estrogen receptor tethered to AP-1 motifs.

569
Except for miR-342, miR-21, miR-143, and miR-145, little is known for the other miRNAs
regarding their roles in breast cancer development. Since our analysis has already
implicated several oncogenic and tumour suppressor miRNAs to be regulated by ER, we
believe that this strategy can provide promising miRNAs candidates for additional
functional exploration.
4. Discussion
Understanding gene regulation is crucial to elucidating the mechanisms of development,
differentiation and signaling response. Over the past three decades, advances in
technologies such as genomic sequencing and expression profiling by microarray have
paved ways to more thorough investigations into gene regulatory networks. These advances
also necessitated the development of bioinformatics. Namely, analytical tools and methods
are continuingly being invented for processing the vast amount of information generated
and mining the corresponding datasets; hence, new discoveries are observed and novel
concepts are developed for hypothesis building and testing. Nonetheless, the accumulation
of datasets sometimes outpaces the development of bioinformatics and a certain amount of
valuable information is left un-mined. In this chapter, we present a case of utilizing
developed bioinformatics tools to learn more about gene regulation network based on
published transcriptional factor binding datasets.
We used a previous published ER ChIP-chip data to find a set of putative ER-regulated
miRNAs. This concept and method can be extended to other aspects. Firstly, several ChIP
based techniques, such as ChIP-PET (paired-end tag), ChIP-DSL (DNA selection and
ligation), were developed to map TFs binding sites. The genome-wide TF binding sites
generated from these variations of ChIP-chip techniques could also be used to map the
miRNAs promoters. Secondly, our methods can be extended to other nuclear hormone
receptors (NHRs) and TFs providing that corresponding genomic coordinates of TFs
binding are available. Importantly, the specificity of TF binding sites could be investigated
by comparing different but related TF binding data. For example, recently by comparison of
ER and estrogen-related receptor (ERR) binding data in breast cancer cell line MCF-7,
Deblois et al. showed that ERR and ER display strict binding site specificity while a small
number of binding sites were shared by both transcriptional factors(Deblois et al.
2009).Another prominent feature of this versatile procedure lies in its easy application and
low cost. In recent years, ChIP based techniques are popular assays to study direct targets of
TFs genome-wide. For example , many NHR binding maps have been published (Deblois
and Giguere 2008). Surprisingly, few miRNAs regulated by a specific NHR were mined
from these valuable datasets. The directly targeted miRNAs by a specific NHR or TF can be
readily discovered through our procedure if the genome-wide binding sites for this TF have
been produced by others. Thus, it avoids redundant experiments and greatly facilitate rapid
discovery.
MiRNAs microarray is a common practice to identify miRNA expression changes upon a
specific treatment. However, there are some limitations inherited in microarray platform.
For instance, microarray data is usually mixed with primary, secondary, and even tertiary
gene expression changes, making it difficult to dissect which TFs are responsible for these
different levels of regulation. Our procedure directly links the candidate TFs with putative
target miRNAs through analyzing ChIP-chip and ChIP-seq binding data. Uniquely, our
analysis also allows investigation into the relationships between mRNAs and miRNAs co-


570
ordinately regulated by a specific TF in a given cell type upon a particular treatment,
providing an entirely new set of information not revealed by mircroarray analysis alone.
However, it should be noted that not all regulatory regions are included in the original
design of ChIP-chip platform. Thus, our analysis can only provide a partial picture that is
dependent on the completeness of ChIP-chip design. As more comprehensive technology
such as ChIP-seq analysis is used in investigation, the genomic coverage will be significantly
improved. Besides, TF binding sites may be located outside of the 50 kb upstream regulatory
region defined in our analysis. Therefore, it is best to complement ChIP data analysis with
microarray studies to obtain comprehensive information on TFs and miRNAs regulation
networks.
5. Conclusion
Understanding the relationships between transcriptional factors and their target mRNAs is
greatly facilitated by genome-wide analysis based on the pairing of chromatin
immunoprecipitation with DNA microarray. However, few miRNAs regulated by
transcription factors have been mined from these data. Our bioinformatics procedure
efficiently utilize genome-wide binding data to screen upstream regulatory regions of all
human miRNAs and hunt for miRNA targets modulated by a specific transcription factor.
As an example, we predicted 59 putative estrogen-responsive miRNAs based on a published
genome-wide ER binding dataset. Several ER-regulated miRNAs were further confirmed in
a breast cancer cell model. Among these, miR-342, miR-21, miR-422a, miR-124, and miR-
181c were generally found to be up-regulated by estrogen treatment; whereas miR-143, miR-
145, and miR-483 were down-regulated. This example demonstrated the power and
efficiency of this novel analysis method. Furthermore, this example also indicated miRNA
target of a specific TF can be equally detected from ChIP-chip based binding data, which are
usually produced for identifying mRNA targets. Integrating our method with routine
analysis procedure will gain a full picture of gene regulation network by simultaneously
elucidating the miRNAs and mRNAs targets of a specific TF.
6. Acknowledgment
We are in debt to Ms. Xuemei Liao for assistance on graphic preparation. The research is
supported by the science foundation of the education department of Henan province (Grant
No. 2011A180009) and a start-up grant from Henan University of Technology (#2009BS040).
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Zaman, M. S., Y. Chen, G. Deng, V. Shahryari, S. O. Suh, S. Saini, S. Majid, J. Liu, G. Khatri,
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Part 8
Gene Expression and Systems Biology

26
Quantification of Gene Expression
Based on Microarray Experiment
Samane F. Farsani and Mahmood A. Mahdavi
Department of Chemical Engineering, Ferdowsi University of Mashhad,
Azadi Square, Pardis Campus, Mashhad,
Iran
1. Introduction
Gene expression is a common process in all forms of living cells including eukaryotes,
prokaryotes and viruses to generate the macromolecular requirements for life. The study of
gene expression provides a systemic comprehension of the cell function for addressing
specific biological questions. This process comprises replication, transcription, RNA
splicing, translation and post translational modification of a single protein. At first, DNA
serves as a template to replicate itself and the production of RNA (transcription), a copy
from the DNA, is mediated by RNA polymerase. In prokaryotes, transcription creates
messenger RNA (mRNA) which doesnt need any additional processing for translation but
this stage in eukaryotes produces a primary transcript of RNA, which needs further
processing prior to becoming a mature mRNA. This step is referred to as RNA splicing that
in the proper context, involves the removal of certain sequences called intervening
sequences, or introns. Hence, the final mRNA contains the remaining sequences, called
exons, which are spliced together (Knapp et al., 1978). In the next stage, so called translation,
mRNA separates from DNA strand and serves as a template for protein production that
such a process is assisted by ribosomes. Proteins are modified after translation in variety of
processes i.e. they are altered at structural level to achieve the final 3D conformation. These
modifications are essential for all aspects of biology and can be performed spontaneously or
driven by enzyme mediation. Common post-translational modifications include
phosphorylation, glycosilation, dimerization or tetramerizaion, etc. (Doyle & Mamula,
2001). Therefore, the transfer of genetic information, from DNA to RNA and to proteins,
ending up with the expression of genes in all cells makes up the central dogma of molecular
biology (Figure 1) (Crick, 1970).
Genomics information is delivered to the cells in three biochemical datasets including the
complete set of mRNA species that result in generating proteins (transcriptomics), the
complete collection of proteins (proteomics), and the complete series of metabolites produced
in the cell (metabolomics) (Figure 2)(Karakach et al., 2010; van der Werf et al., 2005).
Transcriptomics provides a complete profile of RNAs that appear within the cells, tissues and
biological fluids at a specific time. The mRNA levels do vary over time, among diverse cell
types and within cells under different conditions while DNA is more or less unchanged over
the life cycles. Thus, gene expression based on mRNA mediates cellular function and
specifies genes that are turned on or off in different status of cells. As transcriptome


578

Fig. 1. Central dogma of molecular biology.
represents small percentage of the genome and much more complexity, information carried
in the transcriptome has no substantial direct relation to information from the genome (Frith
et al., 2005; Tsiridis & Giannoudis, 2006). Proteomics assists to comprehensively characterize
quantity, structure and activity of the entire complement of expressed proteins (proteome)
in large scale within a cell or tissue at a particular time. In addition, this approach provides
the studies of protein-protein interactions and detailed understanding of the complex
responses of a living system to stimuli (Beranova-Giorgianni, 2003; Hirsch et al., 2004).
However, genome is relatively static while the dynamic proteome changes constantly in
response to environmental signals. This is due to many reasons, including different amino
acid sequences, alternative splicing of mRNAs and post-translational protein modifications
that often give rise to more than one protein per a single gene. Proteomics, therefore,
produce large high dimensional datasets that require powerful tools to handle and analyze
the data effectively (Hegde et al., 2003; Tsiridis & Giannoudis, 2006).

Fig. 2. Biochemical levels of information in gene expression study.
Metabolomics is the study of the entire set of metabolites, low-molecular-weight organic
compounds, in the cell (metabolome) assisting the inference of biological functioning
(Schaub et al., 2009; van der Werf et al., 2005). It involves the large-scale analysis of changes
in metabolites in response to environmental or cellular changes. Metabolomics aims at
quantifying every single metabolite and is one step further than metabolic profiling that
only elucidates an inventory of the metabolites present in the cell (van der Werf et al., 2005).
The transcriptome, proteome and metabolome can change considerably depending on
various environmental conditions and directly represent the status of cellular physiology.
Hence, these sources are so beneficial in understanding biological performance. Although
Genomics Transcriptomics Proteomics Metabolomics

Quantification of Gene Expression Based on Microarray Experiment

579
omics technologies have been advancing over the years, they still contain some drawbacks.
Proteomics and metabolomics offer the holistic and complementary insights into cells
because transcriptomics cannot always reflect corresponding protein or metabolite profiling.
They are, however, limited in lack of standardized methodologies and poor reproducibility
(Pinet, 2009). This is partly due to the heterogeneous characteristics of the compounds
identified. In proteomic analysis, the wide range of proteins makes it difficult to design
standard protocols for identification of compounds. Likewise, metabolomics suffers from
the diverse collection of chemical properties of different metabolites (Karakach et al., 2010).
Scientists are not well trained to cope with the large data and limited availability of
commercial metabolites. Despite these limitations, going from one biochemical level to the
next, information is acquired or lost by regulatory events such as post-transcriptional and
post-translational modifications that occur between these levels. Metabolomics, however, is
valuable as it is the closest to the function of a cell i.e. the phenotype (Tsiridis & Giannoudis,
2006; van der Werf et al., 2005; Zhang et al., 2010).
Compared to proteomics and metabolomics, transcriptomics is a more robust, large-scale,
moderate cost technology of simultaneously measuring thousands of mRNA levels, but
most transcriptomic analysis platforms are not routinely set up to systematically detect
changes in spliced species as nearly 50% of human genes may undergo alternative splicing
(Hegde et al., 2003). Also in some cases mRNA levels are a reasonable proxy for protein
abundance, allowing one to make a rational inference regarding the level of protein
expression based on the levels of mRNA expression. But, sometimes some caution seems
necessary where protein expression is controlled post-transcriptionally by other factors.
Since mRNA molecules are relatively more homogeneous than metabolites and proteins,
and capture methods based on complementary DNA have been developing, the field of
transcriptomics has been more associated with gene expression studies using microarray
technology (Karakach et al., 2010).
In conclusion, the study of omics sciences plays an important role in understanding
different perspectives of cells to gain knowledge about cellular pathways, mechanisms and
functions that eventually make up an expression cycle. The transcriptome is more crucial in
expression measurements while the proteome and the metabolome together assist in
determining the functionality of expressed genes (van der Werf et al., 2005). Thus, effective
integration of omics datasets provides a broader view of systematic changes in expression
levels. However, this integration still remains one of the challenges of systems biology and
functional genomics.
2. Methods to quantifying mRNA level
Composition and differences of various transcriptomes is specified through mRNA level
measurements. There are a number of methods to quantitatively determine this factor
including northern blotting, reverse transcriptase polymerase chain reaction (RT-PCR) and
DNA microarray. These techniques are briefly discussed in the following.
Northern blotting is a standard method for studying the expression profile of specific genes
in mRNA level. It can detect alternatively spliced transcripts and transcript size. In Northern
blot analysis, mRNAs are extracted from sample then separated based on size in gel
electrophoresis (targets). Probes are a complementary sequence to all or a part of interested
mRNAs. Afterwards, targets are transferred to a solid support from an agarose gel to
hybridize with radio-labeled probes. If the probe has complemented sequence to an mRNA,


580
then it will bind to the location of that mRNA on the gel (Trayhuru, 1996). Degree of
radiation gives an indication of expression level in gene of interest. This method is a semi-
quantitative detection because the amount of radioactivity depends to some extent on the
amount of the probe which in turn depends on the amount of mRNA in the sample (Perdew
et al., 2006; Trayhuru, 1996). Northern blotting is an appropriate assay especially for
laboratories which are limited with the lack of specialized equipments and expertise in
molecular biology (Trayhuru, 1996). One of the pitfalls in northern blotting is often sample
degradation through the action of RNases, which can be overcome by proper sterilization of
glassware and reagents and the employment of RNase inhibitors. Also the used chemicals
can be a risk to the researcher.
Polymerase chain reaction (PCR) is an enzymatic assay which produces large amount of a
specific DNA sequence from even a small and complex mixture. Also reverse transcriptase
(RT)-PCR is a rapid and flexible approach for mRNA examination and quantification. In this
method, first the mRNA must be converted to a double-stranded molecule by using the
enzyme reverse transcriptase (Perdew et al., 2006). Since small variations of amplification
efficiencies between samples can result in significant differences in product yield,
quantification of mRNA by RT-PCR is difficult, therefore modified methods have been
developed such as quantitative competitive (QC)-PCR, relative RT-PCR and real time RT-PCR.
The QC-PCR measures the absolute level of a particular mRNA sequence in a biological
sample. It relies on using dilutions of a synthetic RNA called competitors. These competitors
compete with the target cDNA for co-amplification. Since competitor molecule differs in size
from the target one, the two PCR products can be separated by gel electrophoresis. Although
this method provides an accurate result, the design and construction of competitor for each
gene is technically complicated. Validation of the results of the technique is also labor intensive
(Breljak et al., 2005). Relative or semi-quantitative RT-PCR measures mRNA level using a co-
amplified internal control with the gene of interest. Results are reported as ratios of the gene-
specific signal to the internal control signal. Although this method requires only common
laboratory equipment, it suffers from poor dynamic range of the quantification and being time
consuming as well as labor intensive (Lipshutz et al., 1999). A novel approach of PCR, real-
time PCR, is the combination of the best features of both relative and competitive PCR. It is
much faster, higher throughput and less labor-intensive assay than current quantitative PCR.
Furthermore, it combines amplification and detection in one step. Unlike other quantitative
PCR methods, real-time PCR does not need preventing carryover contamination of PCR
products and PCR processing such as electrophoresis. This approach is carried out through
dual labeled fluorogenic probes. The amount of fluorescence emitted is directly proportional
to the amount of product produced in each PCR cycle (Breljak et al., 2005; Heid et al., 1996). In
spite of outstanding advances performed in the area of real-time RT-PCR, competitive and
semi-quantitative RT-PCR may still utilize for relative mRNA quantification especially for
small number of samples (Breljak et al., 2005). RT-PCR is much more sensitive, rapid with a
large dynamic range of quantification. It requires specialized expensive equipment and
ingredient which may be restrictive to some researchers (Perdew et al., 2006; Trayhuru, 1996).
Since undesirable primerprimer interactions may happen, RT-PCR is limited in the number of
genes to be analyzed each time. Some sources of variation such as template concentration and
amplification efficiency make difficult quantification based on RT-PCR (Trayhuru, 1996).
Microarray experiment is an emerging technique as such, based on determining expression
levels of thousands of genes simultaneously. This approach can be considered as a massive


581
parallel Northern blotting. DNA microarray gives a holistic picture of gene expression
within the cell or the sample in different environmental conditions at a specific time (Tarca
et al., 2006). Practically, such high throughput method utilizes an inert surface containing a
certain number of spots. Each spot contains a single species of a nucleic acid representing
the genes of interest (probe). Hybridization between labeled biological sample (target) and
probes creates a signal that represents the level of expression of a gene in a biological
sample. The microarrays have become important because they are easier to use and do not
require large-scale DNA sequencing. However these studies are still limited by lack of
universally accepted standards for data collection, analysis and validation (Bilban et al.,
2002; Russo et al., 2003). Microarrays are quite user friendly and usually consistent with
results produced from northern blotting and PCR; although, these approaches can measure
small levels in gene expression that microarrays cannot. The main advantage of microarrays
is visualizing thousands of genes at a time, while other methods are usually quantifying one
or a small number of genes (Bilban et al., 2002; Trayhuru, 1996).
Some features of the above mentioned methods have been summarized in Table 1.
Regarding the advantages and limitations of each technique, it is concluded that even
though the all methods can measure mRNA levels, they differ on their special attributes.

Method Pros Cons
Northern
blotting
-Detecting alternatively spliced
transcripts
-Detecting transcript size
-Straightforward
-Inexpensive

-Insensitive
-RNase contamination
-Low throughput
-Use of hazardous reagents
-Low quality quantification
-Needs large quantity of RNA
-High background on solid supports
RT-PCR -High sensitive
-Rapid
-Wide dynamic range (Real time RT-
PCR)
-Sensitive and robust
-Nearly high throughput
-needs small sample
-Expensive equipment
-needs expertise in molecular biology
-The ease with which minor
contamination may yield false-positive
results
-Post-PCR manipulation except real time
PCR
Microarray -The parallel quantication of thousands
of genes from multiple samples
-Rapid
-Robust
-Convenient for directed and focussed
studies
-Cost effective
-Easy to use
-do not require large-scale DNA
sequencing
-needs verification
-Difficult to correlate with absolute
transcript number
-Sensitive to alternative splicing
-many factors can affect microarray result:
chip type
sample preparation
data analysis
-Requires bioinformatics for data analysis.
-Lack of standard preprocessing methods
-Low sensitivity of microarray detection
technology
Table 1. Features of conventional techniques to quantifying mRNA level.
Therefore, the selection of methods is performed based on required characteristics in
experimental design. It should be noted that although traditional techniques of gene


582
expression analysis provide valuable biological insights into the living cells, they are
probably limited in some ways such as scale, economy, and sensitivity. As a result,
compared to the other commonly used techniques, quantification based on microarray is
remarkable because of high throughput and cost effective features.
3. Microarray technology
Microarray technology has become one of the most commonly used high-throughput
techniques to query a large variety of biological issues. It enables the simultaneous analysis
of thousands of parameters within one single experiment. Such miniaturized binding
technology is typically divided into DNA, protein, tissue, cellular and chemical compound
microarrays (Templin et al., 2002). Some of the arrays such as protein array and tissue array
will be described in detail with a special emphasis on DNA arrays.
Protein microarrays assist in characterizing of thousands of proteins in a parallel format.
Proteome chips afford researchers a way to address true level of gene function by studying
the pair-wise interactions such as protein-protein, protein-DNA, protein-lipid, protein-drug,
protein-receptor and antigen-antibody (Hall et al., 2007). In this technique, probes such as
aptamers, engineered antibody fragments, affibodies, full-length proteins or protein
domains can be spotted on a microscope slide. The array is then probed with a target
solution and binding detected using the analytical approaches. Antibody microarray is the
most powerful type of protein microarray. Figure 3 shows the detailed view of the steps
taken to carry out antibody microarray experiment (Angenendt, 2005). Tissue microarray
(TMA) technology was developed in order to evaluate the difference of molecular targets (in
the DNA, RNA or protein level) in several thousands of tissue samples at the same time
(Kononen et al., 1998; Singh & Sau, 2010). TMA is constructed from paraffin embedded
material, frozen tissue, paraffin embedded cell lines or cell blocks (Parsons & Grabsch, 2009).
Totally, TMA is made of tissue core samples taken with a precision punching instrument
from donor paraffin blocks. These cores of tissue are arrayed into an empty recipient block,
TMA block (Figure 4). Afterwards, the TMA block is sectioned by using a device called
microtome. The sections are placed on a microscope slide and then analyzed by any
standard histological procedure. From a TMA block, approximately 200300 5-m sections
can be cut and used at independent tests (Parsons & Grabsch, 2009).

Fig. 3. Schematic diagram of an antibody microarray technology.


583
DNA microarray is the most popular type of microarray technology that uses nucleic acid
nucleic acid interactions. It allows measuring the amount of mRNA transcripts for
thousands of genes in different combinations of sample derived from normal and diseased
or treated and non-treated tissues, time courses of treated cells and stages of cell
differentiation or development (Karakach et al., 2010). It has been proved that DNA
microarrays are extremely valuable in studying of expression profiling, sequence
identification and location of transcription factor binding sites (Hall et al., 2007).
4. The DNA microarray experiment
DNA microarrays are currently manufactured using two main techniques: in-situ synthesis
and deposition of pre-synthesized probes (spotted arrays). There are various platforms or
types of DNA microarrays that are commercially available. Figure 5 summarizes some of
these platforms based on different fabrication methods. The two most commonly micarrays
are the affymetrix oligonucleotide chips (Lockhart et al., 1996) and spotted cDNA arrays
(Schena et al., 1995). Experimental steps and construction process of these arrays are
discussed in this section.

Fig. 4. a. Tissue arrayer instrument, b. Extraction of the donor core, c. Insertion into recipient
block (Gulmann & O
Grady, 2003).
4.1 Affymetrix Gene Chips
4.1.1 Fabrication of Affymetrix Gene Chip
The in situ synthesis of oligonucleotides (Affymetrix Gene Chip) can be achieved using a
photolithographic method (Fodor et al., 1991). This approach involves adding of adenine
(A), cytosine (C), guanine (G) and thymine (T) nucleotides step by step through a set of
designed masks. In fabrication process, solid substrate, usually quartz wafer, is washed to
provide uniform hydroxylation of the surface and is then placed in a silane bath. Silane
molecules are capable to directly react with the hydroxyl groups of the quartz. Therefore
starting points are formed to synthesize new oligonucleotide strands. In the following steps,


584
synthetic linkers are attached to silanes and coated with a light-sensitive protecting group
(Figure 6). The first mask is placed over the surface which then exposed to the light source.
Masks selectively direct light toward specific areas on the substrate. Afterwards linker
molecules are activated at the unprotected position. Next, the first of a series of nucleotides,
linked to the light-sensitive agent, is incubated on the surface. Thus, the nucleotides are
chemically coupled to the activated sites. Photo labile agents block further nucleotide
binding to linkers until light subsequently activates them through a new mask. This
chemical cycle is repeated until several hundred thousands of oligonucleotides (probes)
with desired lengths and sequences are synthesized at each of sites on the surface of the chip
(Lipshutz et al., 1999).

Fig. 5. Different microarray platforms and their fabrication methods.


585

Fig. 6. a. Schematic overview on photolithographic fabrication of Gene Chip. b. Drawing of
the lamp, mask and chip (Lipshutz et al., 1999).
4.1.2 Experiment of Affymetrix Gene Chip
Microarrays use various approaches based on uniqueness and composition design rules to
select the 25-nucleotide-length (25mer) probes (Lipshutz et al., 1999). They utilize the Perfect
Match/Mismatch probe strategy (Figure 7). Each gene sequence (or expressed sequence tag
(EST)) is represented by typically 12-20 different probe pairs. The collection of probes for
each gene is referred to as a probeset. Each pair includes a perfect match (PM)
oligonucleotide and a mismatch (MM) oligonucleotide. A PM probe is perfectly
complementary to the gene sequence of interest (Barrett & Kawasaki, 2003; Lipshutz et al.,
1999) while the MM probe has a one-base mismatch in the central base position (the 13th
base). The MM probe is used as an internal control to estimate the signal of any non-specific
hybridization or contaminating fluorescence within measurement (Lipshutz et al., 1999;
Tarca et al., 2006). These probesets are made on array through in situ synthesis and the
microarray will be ready to carry out the experiment.
The basic steps in this single-dye experiment are as follows. Total RNA (or mRNA) is
extracted from the biological sample, called target. The total RNA is then reversed
transcribed to generate double-stranded cDNA. Then, biotin-labeled cRNA is produced
from cDNA using in vitro transcription. Next, biotin-labeled cRNA is fragmented into
smaller segments and hybridized on the array. After a series of washing for removing non-
hybridized material, the array is incubated with appropriate fluorescent dyes linked to the
biotins on the cRNAs. The array is placed in a scanner and emission of uorescent staining
agent is quantified (Schadt et al., 2001). Measurement of the florescent agent intensity
provides an estimate of the level of mRNA within each gene of interest on the chip.
a.
b.


586

Fig. 7. Design of Affymetrix Gene Chip technology.
4.2 Spotted cDNA array
In spotted technology, probe sequences are synthesized separated from the array. In this
technique, the probes correspond to specific genes, expressed sequence tag i.e. a stable
cDNA fragment, or cDNAs from libraries of interest (Bilban et al., 2002). If the quantity of
available probes is limiting, PCR amplification is performed to make sufficient probes. The
PCR products are then analyzed by gel electrophoresis, quantified and eventually spotted
using a robotic printing on the microarray surface. Probes are immobilized or attached at
fixed locations onto the slides electrostatically, through cross-linking by heat or ultraviolet
irradiation and via amines or other active groups on modified slides (Barrett & Kawasaki,
2003). Therefore, the location of each spot on the array can also assist researchers to identify
a desired gene sequence.
Since the cDNA probes are double stranded the array is then heated (or alkali treated) until
the DNA is separated and hybridized to its complementary strand. In this two color
approach there are two samples, a test sample and a reference sample. In order to prepare
the targets, cDNAs are synthesized using reverse transcript of mRNAs in the samples.
Targets are labeled through variety of labeling methods. The most common approach is
labeling with a red and green fluorescent dye, called Cy5 and Cy3, respectively. The labeled
targets are combined and deposited on the array. If a gene is present in one or both samples,
it will bind to its complementary probe according to the complementary base pairing
property of nucleic acids. After washing the array to remove the non-hybridized targets, a


587

Fig. 8. a. Spotted cDNA microarray experiment consists of the following: preparation of
target genes, labeling of the targets, hybridizing, scanning, b. Scanned image of a cDNA
microarray (Karakach et al., 2010).
laser scanner assists to quantify the emission from Cy3 and Cy5 dyes (Figure 8). A green spot
indicates that the corresponding gene is more strongly expressed in the reference sample
compared to the test sample, while a red spot shows the opposite. A yellow spot reveals a gene
in both samples is expressed in the same levels while a black spot shows that the gene is not
express in either sample. The fluorescent spot intensity directly gives an estimate of the
amount of mRNA concentration at specific condition and cell type. Details of each
experimental step have been reviewed elsewhere (Bilban et al., 2002; Karakach et al., 2010).
Characteristics of the two discussed microarrays are summarized in Table 2. This table
provides a comparative view of cDNA and affymetrix oligonucleotide microarrays.
Selection of desired platform is based on biological question which determines aims of the
experiment. In the remainder of the chapter, we will focus mainly on the spotted cDNA
microarray because of limited space even though some of the discussions can be generalized
to other platforms such as affymetrix oligonucleotide array.

Platforms Pros Cons
cDNA microarray
-Low cost
-More flexible
-Easier to customize and analyse
-Wide availability
-No sequence information required
-More variability in system
-Cross-hybridization
-Intensive labour requirement
-Frequent failure of array or
individual spots
Affymetrix
oligonucleotide chip
-More reliable
-Easier to use
- Speed and specific
-Reproducible
-Low failure rate
-Ability to differentiate between splice
variants
-Detection of mutant sequences
-More difficult to analyze
-Expensive array and reagents
-Exact sequence information
necessary
-Lack of flexibility
Table 2. A comparison between cDNA and oligonucleotides arrays.


588
5. Image processing
In the microarray experiment, as mentioned earlier, hybridized slides are inserted into a
scanner to prepare fluorescent images arranged into a matrix of spots. The next step is
processing these images to quantify level of gene expression based on the intensity of each
spot and obtain background estimates and quality measures (Istepanian, 2003, Yang et al.,
2002a). Accuracy of analysis in this phase has remarkable effect on downstream analyses such
as clustering, classification or the identification of differentially expressed genes (Yang et al.,
2001). Generally, laser scanning confocal microscopy acquires fluorescent signals emitted by
fluorescently labelled targets on the array. Scanners detect and record the signals using
photomultiplier tubes (PMT) or charge coupled device (CCD) cameras (Figure 9). These
signals are stored in two 16-bit tiff (tagged image file format) images for further analysis
(Karakach et al., 2010). Images contain information about each fluorescent dye, typically Cy3
and Cy5. Most of the softwares create a composite image by overlaying the two images
corresponding to the individual channels for visualizing different status of genes.

Fig. 9. Photons emit from a fluorescent samples through excitation, enter into a PMT,
resulting in the release of electrons. Analogue signals from PMT are converted into digital
signals by an analog-to-digital (A/D) converter. (More details in Schena, 2003).
Image processing techniques can be divided into the following steps: gridding,
segmentation, quantification and spot quality assessment (Istepanian, 2003; Yang et al.,
2001). Over the last years, a number of commercial and free softwares have been developed
which can perform each step of image processing in a particular approach. These steps are
discussed in more details in following sections.
5.1 Image gridding
The basic layout of a microarray image is determined by the robotic printing devices
(arrayers) as it is known in advance (Figure 10). The arrayer itself consists of a series of pins
arranged as a print tip (also referred to as sub-arrays or grids). The pins pick up reagents
and deposit them on the array. Hence, the spots on array are organized in several print tips
that each one is composed of spots printed with one pin (Karakach et al., 2010). Gridding
(addressing) is the process of finding location of the spots on images. This is carried out
using a simple model based on layout of scanned image. In order to enhance the reliability,
manual intervention is utilized in association with automatic procedures (semi-automatic)
(Yang et al., 2001). However, this can probably make the process very time consuming and
introduce user bias and loss of consistency. At first, the user manually specifies the positions
of spots on the image. Then, a suitable grid pattern is automatically provided from the
indicated positions (Gjerstad et al., 2009; Yang et al., 2002a).
5.2 Image segmentation
Grid spots are partitioned into foreground (within printed spot) and background regions
through a process referred to as segmentation. Foreground pixels represent the true signal
Signal


589
while pixels in the background area correspond to signals not due to hybridization of target
molecules (noise or artifacts) (Yang et al., 2001; Yang et al., 2002a). The most common
segmentation methods are classified based on whether they place restrictions on the spot
geometry. Fixed circle and adaptive circle segmentation methods assume circular spot
shapes, while the histogram and adaptive shape segmentation approaches apply no
restrictions on the shapes of the spots in the estimation of the spot masks. Each
segmentation method generates a spot mask which consists of a set of foreground pixels for
each spot (Karakach et al., 2010; Yang et al., 2001). The simplest method is fixed circle that
assigns a circle with constant diameter to all spots. It characterizes the pixels within the
circle as true signal and the pixels out of the circle as background pixels. Adaptive circles
segmentation estimates the circles diameters separately for each spot (Yang et al., 2002a;
Yang et al., 2001). Since this approach requires the user to adjust spot sizes, it can be time-
consuming for an array with thousands of spots. Furthermore it will be hard to distinguish a
transition between the foreground and background if the signal strength is low (Yang et al.,
2002a). Although most spot shapes are expected to be circular, in practice non-commercial
arrayers rarely print the perfect circular shapes of spots resulting in poor estimates of
uorescent intensities for hybridized targets. Thus, novel approaches known as adaptive
shape segmentation methods has been developed which try to find the best shape of the
spot (Yang et al., 2001; Yang et al., 2002a). These methods are commonly based on the
watershed transform (Beucher & Meyer, 1993) and the seeded region growing algorithm
(SRG) (Adams & Bischof, 1994) which successfully detect different sizes and shapes of
segmented spots (Karakach et al., 2010).

Fig. 10. Common structure of a cDNA microarray slide.
The most widely used method for segmenting spots, without restricting to particular
shapes, is the histogram-based technique (Yang et al., 2001). It defines a target spot mask
whose size is larger than any other spot. Foreground and background intensities of each
spot are estimated from histogram of the pixels within this mask in various ways (Yang et
al., 2002a). This technique directly quantifies values and needs no spot quantification stage.
The discussed segmentation methods are implemented in most softwares to perform
primary level processing of microarray images (Table 3) (Yang et al., 2001).


590
Segmentation method Software Implementing Method
Fixed Circle ScanAnalyze, GenePix, QuantArray
Adaptive Circle QuantArray, GenePix, Dapple, Agilent Feature Extraction
Histogram ImaGene, QuantArray and DeArray
Adaptive shape Spot
Table 3. Different segmentation methods in different image processing softwares.
5.3 Image quantification
After detecting the location of spot and classifying pixels, it is necessary to compute red and
green foreground intensities as well as red and green background fluorescent values for
each spot on the array (Yang et al., 2002a).
5.3.1 Foreground quantification
In fact, the aim of the spot quantification is estimating a quantitative measure which is a
combination of pixel intensity values (Yang et al., 2001). There are different statistics to
compute this measure. Simple sum of pixel intensities is not a good statistic because it
dependent on the size of the spot. Thus, values obtained from spots with different densities
cannot be compared directly. Most microarray imaging softwares estimate the foreground
intensity as the mean or median of pixel values within the segmented spot mask (Yang et al.,
2002a). The median value is more robust to possible outlier pixels; hence it is preferable over
the mean. Also interquartile range (IQR) (i.e., the difference between the 25th and 75th
percentiles) of foreground may be computed for each channel as pixel variation estimation.
5.3.2 Background quantification
Background estimation is generally considered necessary for the aim of performing
background correction (Yang et al., 2002a). Background estimation methods can be classified
into four categories: local, morphological, constant and no adjustment background (Yang et
al., 2001). In the first category, background intensities are computed by focusing on small
regions around the spot mask. Different softwares utilize variety of shapes for these areas
such as square, diamond-shape (referred to as the valley) and circles with different
diameters. Usually, the background measure is estimated by the median of pixel values
within these specific regions; however, it is possible to calculate mean, standard deviation,
and interquartile range of pixels (Yang et al., 2001). Also, there are two types of
morphological filters. The first one corresponds to a non-linear filter called morphological
opening that is obtained by applying a form of local minimum filter (an erosion process)
followed by a local maximum filter (a dilation process) with the same window for each
image (for more details, see (Soli, 1999)). The second one corresponds to a combination of a
closing followed by an opening that removes small dark regions as a better estimate (Wang,
2007; Yang et al., 2001). Constant background is a global method which estimates the mean
or median intensity of the whole image background as a constant background for all spots.
The fourth option is possibility of no background correction (Yang et al., 2001).
5.4 Spot quality assessment
The quality assessment step facilitates to diagnose possible quality problems or even
mistakes that occurred during microarray fabrication and experiment. If this step does not
report any serious irregularities, it will allow performing the following preprocessing steps.


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After calculation of foreground and background intensities, quality measures are estimated
to assess spot quality and reliability. These include variability of pixel values within each
spot, spot area, a circularity measure, relative signal to background intensity (signal to noise
ratio) and flag (Yang et al., 2001; Yang et al., 2002a). Each quality measure can be interpreted
as follows. In most arrays the spots should be of the same size, thus very large or very small
spots may be an indication of problems (Wang, 2007). Eliminating or marking of poor-
quality and low-intensity spots is called flagging. This is zero if the spot is good, but will
take different values if the spot has problems. Different image processing software uses
different flag values for different problems, but the typical flagged spots are:
1. Bad spot: The pixel standard deviation is considerably higher than the pixel mean.
2. Dark spot: The signal of the spot is very weak.
3. Negative spot: The signal of the spot is less than the background value.
4. Manually flagged spot: The user has flagged the spot using the image processing
software (Stekel, 2003).
Performing the four steps of image processing, quantitative parameters are generated in an
output file of the software as shown in Table 4. These measures exhibit some of the location
information, foreground and background quantifications and quality measures in a
microarray experiment.
6. Preprocessing of cDNA microarray data
Prior to identification of DEGs, the data collected from image processing step needs to be
preprocessed. This important step in microarray data analysis removes non-biological
variations, makes data more meaningful, transforms data into an appropriate scale for
analysis and enhances the quality of subsequent analysis. There are a number of approaches
for preprocessing such as background correction, logarithm transformation and
normalization of microarray data. It should be mentioned that spot quality assessment
(section 5.4) in image processing could also be considered as a preprocessing step.
6.1 Background correction
Background correction is a necessary step in preprocessing of cDNA microarray data since
the quantified uorescence intensity of a spot contains background noise which does not
reflect the true hybridization of the target to the probe. Background noise results from
several sources such as non-specific hybridization of labeled target to the array surface,
autouorescence from the array surface or detection instrument, spatial heterogeneity across
the arrays (Ritchie et al., 2007; Tarca et al., 2006). For the purpose of background correction,
it is conventionally assumed that the background signals are additive to the foreground
signals (Ritchie et al., 2007). Also, the standard approach for correction is subtracting an
estimate of the local background intensity from the foreground intensity. Despite spread
implementation of this approach in different software packages, it may cause problems. It
generates negative corrected intensities resulting in missing log ratios, if the background
intensity is larger than the foreground intensity. Even when there is no missing, it results
highly variable log-ratios for low intensity spots (Kooperberg et al., 2002). Also it may cause
some difficulties in the identification of differentially expressed genes (Yang et al., 2001). To
overcome aforementioned limitations, alternative approaches have been proposed such as
subtractive correction using an estimate of the global instead of the local background and


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Index grid.r grid.c spot.r spot.c Area Gmean Gmedian GIQR bgGmean
1 1 1 1 1 95 22028.26 23219 0.564843 372.6964
2 1 1 1 2 85 25613.2 20827 0.672128 928.8974
3 1 1 1 3 77 22652.39 17498 0.939413 1371.86
4 1 1 1 4 21 8929.286 5270 1.975485 250.5417
5 1 1 1 5 21 8746.476 7396 2.518724 262.0417
6 1 1 1 6 112 37010.08 41539 0.943238 499.1722

bgGmed bgGSD Valley morphG morphG.erode morphG.close.
open
Logratio Perimeter Circularity Badspot
307 0.252131 306 182 153 289 -0.17171 40 0.746128 0
299 0.390198 280 171 153 278 -0.16341 36 0.824183 0
339 0.820078 275 153 136 278 -0.15408 34 0.837033 0
244 0.270411 258 153 132 271 0.80675 16 1.030835 0
235 0.275412 244 153 132 216 -0.10662 16 1.030835 0
304 0.740031 244 139 120 224 -0.44679 44 0.72698 0
381 1.05041 243 138 120 224 -0.21073 34 0.739198 0
Table 4. Partial output file from Spot software for green (Cy3) channel: Location information
( spot index, grid row, grid column, spot row and spot column), Area: the number of
foreground pixels for each spot, Gmean: the average of foreground pixel values, Gmedian:
the median of foreground pixel values, GIQR: the interquartile range of foreground pixel,
bgGmean: the average of background pixel values, bgGmed: the median of background
pixel values, bgGSD: the standard deviation of background pixel values , valleyG: the
background intensity estimate from the local background valley method, morphG:
background estimate using morphological opening, morphG.erode: green background
estimate using morphological erosion, morphG.close.open: green background estimate
using morphological closing-opening, Logratio: the log-ratio for each spot is calculated
as
2
2
log ( )
log ( )
Rmean bgmedR
Gmean bgmedG
, Circularity: Shape of spot defined as

2
4 Area
primeter

, Badspot: If the
spot has problem, it equals to 1, otherwise 0.
morphological opening filters which provide less variable log ratios (Ritchie et al., 2007;
Yang et al., 2001). Some methods utilize statistical models, other than subtraction, to adjust
the background estimate. A simpler background correction method was proposed to avoid
negative corrected values. This model adjusts the foreground intensities by subtracting the
background when the difference between the foreground and background is larger than a
threshold value. However, when the difference is less than the threshold, subtraction is
replaced by a smooth monotonic function. Kooperberg et al., 2002 proposed an empirical
bayes model to correct background noise. A remarkable feature of this method is only the
use of the mean, median and standard deviation statistics for each spot that are provided
through the scanning software. In other methods, the models based on variance stabilizing
transformations were proposed for incorporating additive components which prevent
negative intensities. The Models use an arcsinh function instead of the logarithm
transformation of the data. Also background correction and normalization are
simultaneously performed on all the arrays together (Kooperberg et al., 2002; Ritchie et al.,
2007). It is notable that no background correction has been recommended. Sometimes, local


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background methods show greater variability around the low intensity spots rather than no
background adjustment (Yang et al., 2001).
6.2 Logarithm transformation
Before normalization, a logarithmic transformation is often performed on microarray data.
This transformation is successful at reducing some of the variations, and makes the
multiplicative noise of the data additive. Also data is transformed into a symmetrical and
normal data distributed around zero through taking log transformation. This means that up-
and down-expressed genes are treated in identical way (Quackenbush, 2002). However, the
log transformed ratios limit subsequent analyses and the amount of information gained from
the data (Zhao et al., 2007). The ratios do not provide information about the absolute
expression levels. Also, the use of the ratios remarkably dependents on the choice of the
reference sample, which is uncharacterized and not accurately reproduced. This will make it
difficult to compare between data sets that use different reference samples (Zhao et al., 2007).
6.3 Normalization
There is variety of variations from the beginning of the experimental process through
generation of raw data in microarray experiment. Two sources of variations are biological
variations and procedural variations. Biological variations are the consequence of
environmental changes or biological differences of the studied genes on the array. These are
desired variations and represent the true changes in expression cycle. Procedural variations
can be attributed to many sources such as microarray fabrication, mRNA preparation, reverse
transcription, labelling, amplification, pin geometry, fluctuations in target volumes, target
fixation, hybridization parameters, overshining, and image analysis. Detail description of each
variation source is presented elsewhere (Schuchhardt et al., 2000; Yang et al., 2002b).
Procedural variations can be removed (or minimized) using statistical approaches, so that
biological variations are more accurately detected. The processes and transformations for the
purpose of adjusting data are referred to as normalization. Hence, normalization is a crucial
step in microarray data preprocessing, since data interpretation and identification of DEGs
depends on the choice of normalization method (Yang et al., 2002b).
Different biases arise from variations in the microarray data. The most common is dye bias
i.e. imbalance between the two channels due to differences between physical properties of
dyes and detection efficiencies between the fluorescent dyes. Other biases such as print tip
bias and spatial bias may arise from variation between spatial positions on the array due to
differences between the print-tips on the arrayer (Smyth & Speed, 2003). In order to remove
biases, numerous normalization approaches have been proposed. These algorithms can be
applied either globally to an entire data set or locally to a subset of the data. For cDNA
spotted microarray, local normalization is often applied to each print tip (Quackenbush,
2002). Normalization methods can be divided into two main categories: within-array
normalizations and between-array normalizations. Within-array normalization has to be
performed to adjust procedural variations for each single microarray. Some of more
common approaches are as follows.
6.3.1 Global normalization
Global normalization is the simplest and most common within-array normalization method.
It assumes the red and green intensities are related by a constant factor k, namely R=kG. The


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log-ratios are corrected by subtracting a constant c to get normalized values. (log , log ) R G
are background corrected red and green intensities and then:

2 2 2 2
log ( ) log ( ) log ( ) log ( )
i i i
i i i
normalized
R R R
c k
G G G

= =

(1)
The global constant c is usually estimated from the mean or median log ratios over a subset
of the genes assumed to be not differentially expressed, although variety of strategies have
been proposed for estimating this global constant (Quackenbush, 2002; Smyth & Speed,
2003). Global method is limited in adjusting intensity-dependent dye bias and spatial bias.
6.3.2 Intensity-dependent linear normalization
In most cases, the dye bias appears to be dependent on spot intensity linearly or nonlinearly.
Linear normalization assumes the relation between M and A is linear based on model
0 1
M A = +
, where
0 1
( , ) can be estimated by least squares estimation. The most common
method to visualize behavior of two channels is MA plot which uses log intensity ratios (M)
and log intensity averages (A) where M and A are usually defined for each gene as
2
log ( )
i
i
i
R
M
G
= and
2
1
log ( )
2
i i i
A R G =
6.3.3 Intensity dependent nonlinear normalization
The most efficient and widely used nonlinear normalization approach was proposed by
Yang et al., 2002b. It considers the relation between M and A as a function of A i.e. M = c(A),
instead of a linear relation. The estimation of c(A) is made by using a loess (locally weighted
scatter plot smoother) function to operate a local scatter plot smoothing to the MA plot. The
scatter plot smoother performs local linear fits in overlapping windows on the data and then
combines the regressions to produce a smooth curve. This method can be divided into three
categories based on the type of the treatment performed on the data. These categories
include: global loess, print tip loess, and two-dimensional loess. Global Loess (Gloess)
normalization method uses the loess function to perform a local A-dependent analysis:

2 2 2 2
log ( ) log ( ) ( ) log ( ) log ( ( ))
i i i
i i i
normalized
R R R
c A k A
G G G

= =

(2)
Where c(A) is the loess fit to the MA plot for all printed genes (Smyth & Speed, 2003).
Print tip loess (PTloess) is performed within each of the print tip groups separately as
follows:

2 2 2 2
log ( ) log ( ) ( ) log ( ) log ( ( ))
i i i
p p
i i i
normalized
R R R
c A k A
G G G

= =

(3)
Where ( )
p
c A is the loess fit as a function of A for the
th
p print tip. By fitting separate loess
lines for each group and correcting the intensity by its corresponding loess lines, not only
the dye bias will be removed, but it can also correct the print tip bias. Two dimensional loess
(twoDloess) method fits a smooth two-dimensional surface to the data which is a function of
overall row position r and column position c of the spot on the array. The intensity-based


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trend is assumed to be global rather than varying across the array as for print-tip loess
normalization.

2 2
log ( ) log ( ) ( , )
i i
i i
normalized
R R
loess r c
G G

=

(4)
Where loess(r,c) is a loess fit calculated based on the position of the spots. The three
techniques remove different biases arose from the experiment. Gloess removes the dye bias
dependent to spot intensity. PTloess removes spatial bias introduced from print tips and
twoDloess removes the spatial bias on the overall slide.
The above normalization methods are applied to a single microarray. But in order to be able
to facilitate comparison and integration of different microarrays, it is required to remove the
variability caused by using multiple microarrays. It can be performed through the following
approaches. Differences between arrays may arise from differences in print quality or from
differences in ambient conditions when the plates are processed (Smyth & Speed, 2003).
6.3.4 Scale normalization
This method is a simple scaling of the data on multiple arrays so that each array has
identical median absolute deviation (MAD). It aims to remove scale differences in the data
and assumes that the log ratios on the array follow a normal distribution with mean zero
and variance
2 2
j
a where
2
is the variance of the true log ratios and
j
a is the scale factor
for array j with n denoting the total number of arrays (Yang et al., 2002b).

1
n
n
j j j
j
a MAD MAD
=
=

(5)
Where
j
MAD denote the median absolute deviation for array i. Then

{ }
( )
j j j j
MAD median M median M = (6)
Finally, all log ratios are scaled through dividing by the same scale factor for each array. It is
notable that scale normalization can also be applied to data within a microarray locally at
the print tip level.
6.3.5 Quantile normalization
Quantile normalization was initially developed for the Affymetrix single channel chip, and
then extended for two color cDNA microarrays. The goal of this method is to produce the
same empirical distributions of expression levels on all arrays analyzed. It relies on the
assumption that the probe intensities among arrays are always exactly the same, regardless
of biology or study design. Clearly the situation where all samples have equal amounts of
expressed genes is the exception, not the rule, making it the rare case where quantile
normalization will normalize data without introducing errors. Quantile normalization is
carried out through the following steps: Suppose that we have the (log base 2 transformed)
probe level expression values from p genes and n arrays in a p n matrix
{ }
1 j
X X = with
i = 1,2,,p and j = 1,2,,n. First, each column of X separately is ranked to generate a p n


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matrix
{ }
1 j
Y Y = . Next, the average of each row of Y is computed and generated
m
X .
m
X is
assigned to each column of Y to get a matrix denoted as
sort
X . Finally, the normalized genes
for each array is provided by rearranging each column of
sort
X to have the same ordering as
the corresponding column of the matrix X so that empirical distributions of the normalized
genes are the same across arrays. Because the algorithm consists of only sorting and
averaging operations, it runs quickly, even with large data sets (Bolstad et al., 2003). (More
details in (Stafford, 2008))
All above normalization methods utilize certain critical biological and statistical
assumptions about data distribution which may not be valid in practice. The main
assumption is that the most genes on the array are non-differentially expressed between the
two samples and the number of up-regulated genes approximately equals the number of
down-regulated genes. In such cases, above-mentioned normalization methods may yield
unreliable results. Xiong et al., 2008 proposed a novel statistical method based on the
Generalized Procrustes Analysis (GPA) algorithm free of assumption (Xiong et al., 2008).
7. Differentially gene expression
Once the data is normalized, further analysis is necessary to obtain biologically meaningful
results. In fact, the main purpose of microarray experiment is to identifying genes that are
significantly differentially expressed under different biological, and/or clinical conditions.
A growing number of approaches have been presented to fulfill this purpose that can be
divided in three categories: marginal filters, wrappers, and embedded methods. The
wrapper and embedded methods are a type of search algorithms by which subsets of genes
that are useful to define a good predictor are generated. Evaluation of a specific subset of
genes is provided by running a specific classification model on the subset. The filter
approaches are scalable and fast methods and independent of the classification algorithm
including t-tests and nonparametric tests and analysis of variance (ANOVA) (Saeys et al.,
2007). We will provide a brief overview on some of the popular statistical differential
expression methods. It is notable that various methods usually identify different ordered list
of significant genes since each approach is based on a specific set of assumptions, and takes
certain features of dataset into account.
Fold Change (FC) cutoff is one of the early approaches for DEG identification that is still
widely used to rank genes in microarray assays. In this method, when ratio of two color
intensities from each gene exceeds a pre-set threshold is said to be differentially expressed.
Usually a threshold of twofold up- or down-regulation is considered as cutoff value in most
biological studies. This method ranks genes based on the ratio of average gene expression
under two different groups or conditions. Simplicity is a main reason for popularity of fold
change approach. Also a major drawback is that it does not consider variance of the
expression values quantified. Hence, in order to cope with this problem, it will be used in
combination with other statistical methods (Tarca et al., 2006). There also exists variety of
statistical tests instead of using a fold change cutoff, for a correct selection of deferentially
expressed genes. A simple but popular method is the t-test and its variants (Cui & Churchill,
2003). The t-test performs according to the simple estimation of the population variance for
a gene through the sample variance of its expression levels. It typically compares the
difference between the mean expression levels among the two groups, considering the
variability of genes in their ranking (Tarca et al., 2006). T-test depends on the type of


597
distribution of the gene expression data. Thus, may not properly perform when data exhibit
a strong departure from the normal distribution. Also the performance of t-test will be poor
when sample sizes are small, because variance estimation is more challenging (Yan et al.,
2005).
The ANOVA approach is a generalization of the t-test that can be used when more than two
conditions are compared. The idea underlying ANOVA is to make a model that considers
the variation sources that affect measurements. Then variance of each individual variable in
the model is computed using expression data (Tarca et al., 2006). In order to improve the
performance of the ordinary t-test and produce more stable results, modified t-statistics are
alternatively proposed. The main difference between an ordinary t-statistic and these novel
statistics is that the latter estimate variability regarding to information not only from the
gene tested, but also from other genes displaying a similar magnitude of expression level
(Smyth, 2004). Two commonly used approaches, i.e. the modified t-statistic methods
(empirical Bayes and SAM), will be described in more detail as fallows.
7.1 The empirical Bayes t-test LIMMA
This empirical Bayes t-tset has been implemented in the limma R statistical package. In this
approach, gene-wise linear models are separately made to represent the design of a
microarray experiment. Next, the coefficients of each linear model are estimated through the
expression data. After quantification of coefficients of model and standard errors, moderate
t, F and B (log-odds) statistics of differential expression are computed using empirical Bayes
approach. It is equivalent to reduction of the gene-wise sample variance towards a pooled
estimate producing more stable result when the number of measurements is small in
experiments. Finally, genes can be ranked based on one of the chosen statistics. A more
detailed derivation can be found in (Smyth, 2004).
7.2 Signicance analysis of microarrays (SAM)
SAM is a statistical technique, proposed by Tusher et al., 2001. It utilizes a non-parametric
statistics, since the expression data may not be normally distributed. Modified t-statistic
used in this method is essentially similar to the moderated t-statistic used in limma but have
no associated distributional theory. Also the empirical bayes method provides a more
complex model of the gene variance. SAM assigns a score to each gene based on change in
gene expression relative to the standard deviation over repeated measurements for that
gene (Smyth, 2004). Genes with scores greater than a threshold are considered differentially
expressed. The threshold significance is determined by the user based on the FDR. The
proportion of such genes identified by chance (false positives) is the false discovery rate
(FDR). To estimate the FDR, nonsense genes are specified using random permutations of the
repeated measurements (Tusher et al., 2001; Yan et al., 2005).
8. R and bioconductor packages
Microarray experiments produce large and highly complex datasets. Access to an efficient
statistical computing environment is a critical aspect of the analysis of these gene expression
datasets. There are a lot of free and commercial software. In most cases, the microarray kits
come with the software that adequately analyses microarray data. One of the best options
for data analysis is the R statistical programming environment (www.rproject.org) where


598
the open-source Bioconductor R packages (www.bioconductor.org) are resourceful and
effective in dealing with these microarray data.
There are plenty of packages such as limma, marray and arrayQuality for two-color spotted
arrays or affy, affyPLM, affyPara and gcrma for Affymetrix array and Agi4x44PreProcess
and AgiMicroRna for Agilent chips. The complete documentation of Bioconductor packages
can be found on the Bioconductor project web site at: http://www.bioconductor.org/help/
bioc-views/release/bioc/. Bioconductor packages remove noise from measurements of
microarray experiments through preprocessing of data. Also they specialize in various
related tasks in handling microarray data. Some packages are dedicated to facilitation and
automation of array data input and applied to detection of spatial and dye effects on arrays
via a variety of diagnostic plots and graphs. In addition to the primary fluorescence
intensity data, these packages also extract textual information on probe sequences and target
samples, such as gene annotations, layout array, target sample descriptions and
hybridization conditions, etc.
Limma package implements tools for data quality assesment, background correction,
normalization and identification of DEGs in microarray experiments. Marray package also
provides alternative functions for reading microarray data into R, normalization data and
diagnostic plots of different measurements. Limma and marray packages share some
features (Smyth & Speed, 2003; Yang et al., 2002b).
In the following, in a case study, we will demonstrate a microarray analysis flow using
Bioconductor R packages in experimental design, data preprocessing, and differential
expression detection. This analysis is performed using Bioconductor Release 2.7 based on R
Version 2.9.
8.1 Step by step microarray analysis
The publicly available dataset from Swirl zebra fish two-color spotted microarray
experiment was used as a typical example in this analysis. Swirl is a point mutant in the
BMP2 gene that affects the dorsal/ventral body axis. In this experiment, two sets of dye-
swap were prepared. On the first array, the wild type and mutated samples were labelled
with Cy5 and Cy3 dyes, respectively. On the second array, the Cy5 and Cy3 dyes were
swapped for the samples. The next two arrays were replicates of the first two arrays,
respectively (Table 5). Thus, four arrays were prepared. Each array consisted of 16 print tips
(4 by 4) and each print tip comprised 22 by 24 spots. Therefore, each array accounted for
8448 spots. Once the experimental steps were carried out, each array was scanned and then
analyzed by SPOT software (Buckley, 2000). The main purpose of the Swirl experiment is to
find genes with altered expression in the swirl mutant compared to wild type zebra sh.

Date FileName Slide number Conditions
2001/9/20 Swirl.1.spot 81 Swirl(Cy3), wild type (Cy5)
Table 5. All experiments for the study of Swirl mutant.


599
In order to analyze the microarray data, a directory of all the image processing output files
should be created (.spot les). This directory includes a file containing experiment
description (SwirlSamples.txt file) and a file describing information on probe sequences,
such as gene names, spot ID (fish.gal). Then R is started in the desired working directory.
The following command will load Limma and marray packages for preprocessing swirl
data.

>library (limma)
>library (marray)

Information about the hybridizations and the raw uorescent intensities data are provided
through the following commands

>targets <- readTargets (SwirlSamples.txt)
>RG <- read.maimages (targets$FileName, source=spot)

In order to identify gene names the following command may be used,

>Genes <- readGAL(fish.gal)

and the layout information of slides uses this command,

>Layout <- getLayout (Genes)

Qualitative assessment of arrays can be performed using different plots and graphs in
microarray experiments. Therefore, serious quality problems and sources of artifacts will be
identified in the data. In this step, the background signal, different biases such as dye bias
and spatial bias are evaluated using visualization techniques. According to the results of the
quality assessment, the need for each preprocessing method is clearly revealed. Firstly, the
background signal distribution is evaluated to identify whether there is any region with
non-uniformity distribution.

>imageplot (log2(RG$Rb[,1]), Layout, low="white", high="red")
>imageplot (log2(RG$Gb[,1]), Layout, low="white", high="green")

Figure 11 shows that the background signals in both red and green channels are unreliably
high in some region of array. It can be concluded as that there is spatial non-uniformity.

Fig. 11. Image of green and red channel background intensities for slide 1.


600
Therefore background correction is performed on swirl data. Since data have no negative
value, subtractive methods can be carried out. Background corrected M and A-values are
generated through subtraction method as follows

>MA <- normalizeWithinArrays (RG, method=none)

Secondly, we visualize the intensity range of M-values for each individual microarray using
MA-plots. The signals include both background signals and foreground signals. These plots
are generated using the following commands:

> plotMA (MA[,1], main="slide 1", ylim=c(-3,3))
> plotMA (MA[,2], main="slide 2", ylim=c(-3,3))

Figure 12 shows MA-plots of raw data of two slides of swirl experiment. Swirl experiment
satisfies major assumption in microarray experiment i.e. a small percentage of genes are
expected to be differentially expressed. Therefore, the majority of the points on the y axis
(M-value) would be located at 0, since log (1) is 0. The shape of the curve on slide 1 shows
more nonlinear dependence on the overall spot intensity than slide 2. Therefore,
normalization will attempt to remove the curvature of the spread and centralize the data
around zero axis.

Fig. 12. MA plots for slide 1 and slide 2 in swirl experiment.
Another diagnostic plot is boxplot which can be useful for comparing M-values and
homogeneity between print tip group and slides. Boxplot in different print tip is plotted
using marray package after generation background corrected data by maNorm function.

>swirl.norm <- maNorm (swirl, norm="none")
>boxplot (swirl.norm[,1], xvar="maPrintTip", yvar="maM", main="slide
1")

The following command also produces a boxplot of the prenormalization M-values for all
four arrays in the swirl experiment.

>boxplot (MA$M~col (MA$M), xlab="slides", ylab="M")


601
A boxplot shows graphically 5-number summary of data, the median, the upper and lower
quartiles, the range, and individual extreme values. The central box in the plot represents
the interquartile range (IQR), which is specified as the difference between the 75th percentile
and 25th percentile. The width of a box represents the variability of the data and solid line in
the middle of the box represents the median. Extreme values, greater than 1.5IQR above
the 75th percentile and less than 1.5IQR below the 25th percentile, are plotted individually
(54).

Fig. 13. Boxplots in different slides and print tips of slide 1.


602
In the next step, we can normalize data through different within and between array
normalization using both marray and limma packages. The prenormalization MAplot and
boxplot for slide 1 in Figures 12 and 13 illustrate the nonlinear dependence of the M-value
on the overall spot intensity A and the existence of spatial biases. We thus perform PTloess
normalization on this data. In the following scale normalization will be performed on the
swirl data because four slides have different spread of M-values (Figure 14).

Fig. 14. Swirl data after PTloess normalization and PTloess fallowing Scale normalization.


603
>MA <- normalizeWithinArray(RG)
>MA <- normalizeBetweenArrays (MA, method=scale)

The closer the solid line to zero line in Figure 14 the more centrality of the data after
normalization. In boxplot plotted between arrays when the width of the rectangles are
approximately the same the distribution of the spots on replicate arrays are the most similar
that means between-array normalization method has been selected appropriately.
In order to detect genes with differential expression between wild type and mutant samples,
linear model and empirical bayes methods in limma package are used (Smyth, 2004). Dye
swap samples are specified using the design matrix, which allows calculating of the average
M- values across multiple arrays.

>design <- c(-1,1,-1,1)

M-values are estimated between these two samples using the lmFit function.

>fit <- lmFit(MA,design)

Moderated t-statistics and logodds (B-statistics) of expression data are calculated using
empirical Bayes methods

> fit <- eBayes (fit)

A summary table of some statistics for the top genes will be obtained using the following
command.

>topTable(fit, number=10, adjust=fdr, sort.by="t")

ID Name M-value Moderated-t B Adj.P.val
Control BMP2 -2.205288 -21.06952 7.960750 0.0003572816
Control BMP2 -2.296045 -20.28697 7.778330 0.0003572816
Control Dlx3 -2.184900 -20.01066 7.710959 0.0003572816
Control Dlx3 -2.180471 -19.63599 7.710959 0.0003572816
fb94h06 20-L12 1.271119 14.08467 7.617005 0.0020666932
fb40h07 7-D14 1.347207 13.52924 5.535983 0.0020666932
fc22a09 27-E17 1.266129 13.41339 5.483567 0.0020666932
fb85f09 18-G18 1.275686 13.39543 5.475386 0.0020666932
fc10h09 24-H18 1.195126 13.23722 5.402676 0.0020666932
fb85a01 18-E1 -1.287128 -13.07059 5.324819 0.0020666932
Table 6. Top 10 genes from the Swirl data.
The moderated t-statistic with adjusted p-values can identify differentially expressed genes.
As seen in Table 6, it can sort both copies of the gene BMP2 knocked out and both copies of
Dlx3, which is a known target of BMP


604
9. Conclusion
Gene expression is a common process in all forms of living cells to generate the
macromolecules which are necessary for life. Systemic comprehension of the cell function is
provided using study of gene expression. Investigation of molecular dynamics of the cell
can be performed in three biochemical levels, transcriptomics, proteomics, metabolomics.
Compared to others, transcriptomics is a more robust, large-scale, moderate cost technology
of simultaneously measuring thousands of mRNA level. There are various techniques for
quantifying gene expression based on mRNA. However gene expression traditional
techniques provide valuable biological information, they are limited in some ways such as
scale, economy and sensitivity. Therefore, compared to the other commonly used
techniques, quantification based on microarray is really remarkable because of being high
throughput and cost effective. It enables the simultaneous analysis of thousands of genes
within one single experiment. Such miniaturized binding technology is typically divided
into DNA, protein, tissue, cellular and chemical compound microarrays. DNA microarrays
are the most popular type of this technology which currently manufactured through two
main approaches: in situ synthesis and deposition of pre-synthesized probes (spotted
arrays). We focused mainly on the spotted cDNA microarray. After microarray experiment,
slides are inserted into scanner. The output data are fluorescent images arranged into a
matrix of spots. Then, images are processed to quantify level of gene expression based on
the intensity of each spot and obtain background estimates and quality measures. It is
performed in gridding, segmentation, quantification and spot quality assessment stages. The
output data from image processing stage needs to be preprocessed to eliminate non-
biological variations, transform data into a suitable scale and improve the quality of
downstream analysis. These are performed using background correction, logarithm
transformation and normalization of microarray data. Finally, identification of genes that
are significantly differentially expressed under different conditions can be carried out using
marginal filters, wrappers, and embedded methods. We pointed some of the filter
approaches such as t-test and its variants such as moderated t-test and SAM approach and
analysis of variance (ANOVA). In summary, in order to analyze microarray data, the R
statistical programming environment is chosen where the Bioconductor R packages such as
limma and marray are effective in processing these microarray data. These packages address
data input, production of diagnostic plots to detection of different biases, the statistical
methods of removing experimental noises and errors on the spots within and between
arrays. Finally, limma package is also used as a powerful tool to identification of
differentially expressed genes.
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27
On-Chip Living-Cell
Microarrays for Network Biology
Ronnie Willaert and Hichem Sahli
Vrije Universiteit Brussel,
Belgium
1. Introduction
The recently developed field of systems biology creates a new framework for understanding
the molecular basis of physiological or pathophysiological states of cells. Screening
modalities that can be used on single cells are needed to study cellular systems biology. The
recent development of cellular microarrays has provided a method for the complex
molecular analysis of living, single cells (Chen & Davies, 2006). Unlike other high-
throughput systems, such as gene expression profiling microarrays or protein microarrays,
cellular microarrays use a printed pattern of geographically distinct spots to probe living
cells, rather than cell lysates, or other non-viable samples. Among the most powerful tools to
assay gene function on a genome-wide scale in the physiological context of intact living cells
are fluorescence microscopy and related imaging techniques (Pepperkok & Ellenberg, 2006).
To enable these techniques to be applied to functional genomics experiments, fluorescence
microscopy is making the transition to a quantitative and high-throughput technology.
The combination of time-lapse microscopy, quantitative image analysis and fluorescent
protein reporters has enabled observation of multiple cellular components over time in
individual cells (Locke & Elowitz, 2009). In conjunction with mathematical modelling,
these techniques are now providing powerful insights into genetic and proteomic
behaviour in diverse microbial systems. Recently, a quantitative system-wide analysis of
mRNA and protein expression in individual cells with single-molecule sensitivity using a
yellow fluorescent protein fusion library for E. coli has been realised (Taniguchi et al.,
2010).
2. Microfluidic chips for cell microarrays
2.1 Cell assays and cell microarrays
A cell assay is defined here as a measurement and analysis of the cellular response, at a
given level, to a chemical and/or physical stimulus (Barbulovic-Nad & Wheeler, 2008).
Cellular responses are diverse, e.g. alterations of intracellular and extracellular
biochemistry, cell morphology, motility and (de)adhesion, survival and apoptosis, and
proliferation properties. These responses characterise single aspects of cell phenotype, and
are typically monitored in a culture dish or a multiwell plate, while more recently
microfluidic devices have been employed. While culture dishes require millilitre volumes of
media and reagents, multiwell plates contain microliter volumes and enable simultaneous


610
analysis of multiple cell types or stimuli. Experiments with multiwell plates are typically
integrated in a robotic analysis platform. Two major drawbacks of robotic platforms are the
expense of the instrumentation, and the cost of experimental consumables.
The use of microarrays was first reported in 1989 (Ekins et al., 1989). The variety and
diversity of microarrays has become impressive. Three main types of microarrays have been
developed: DNA microarrays, protein microarrays, and cell microarrays (Barbulovic-Nad et
al., 2006). Several different approaches to cell microarrays have been explored to investigate
gene expression, cell-surface interactions, extracellular matrix composition, cell migration
and proliferation, the effects of drugs on cellular activity and many other areas (Angres,
2005). There are two fundamental methods to produce cell microarrays: the indirect and the
direct method. The indirect method i.e. the reverse transfection method was developed
by Ziauddin and Sabbatini (2001). In the direct method, the cells are printed onto a
substrate. In few cases contact-based microarrayers are used, but more often non-contact-
based devices are used.
Miniaturisation of cellular assays via cell microarrays increases assay throughput while
reducing reagent consumption and the number of cells required, making these systems
attractive for a wide range of assays in drug discovery, toxicology, and stem cell research
(Fernandes et al., 2009). Cell microarrays have been developed for highly parallel, high-
throughput analyses of cell phenotypes (Narayanaswamy et al., 2006), assessing cell
proliferation and morphology (Bochner et al., 2001; Xu, 2002), protein expression levels
(Schwenk et al., 2002), and imaging of tissues (Kononen et al., 1998; Radhakrishnan et al.,
2008) and single cells (Biran et al., 2003). In these initially developed living-cell microarrays,
microbial cells were printed on an agar growth medium and could grow as microcolonies,
or cells were grown in multiwell plates and printed on a glass slide for imaging, or only
short time analyses on living cells were performed. High-throughput experiments on a
library of cells require on-chip cell culture. Microchip 2- or 3-D cell cultivation techniques
can provide many advantages for cell culture systems because the scale of the cultivated
environment inside the microchip is fitted to the size of the cells. Table 1 gives some
examples of developed mammalian cell microarrays/wells.
2.2 Cell assays in microfluidics
Microfrabrication technology originated from the electronics industry, where 3D micro-
features for electronic devices were manufactured in the sub-centimeter to sub-micrometer
range using lithography techniques (Franssila, 2010). Microfluidics emerged as an extension
of MEMS (Micro Electro Mechanical Systems) technology at the beginning of the 1980s.
Microfluidics is a technology that is characterised by devices containing networks of
micrometer-dimension channels (Whitesides, 2006). It involves the manipulation of very
small fluid volumes, enabling the creation and control of l to nl volume reactors.
Microsystems create new opportunities for the spatial and temporal control of cell
proliferation and stimuli by combining surfaces that mimic complex biochemistries and
geometries of extracellular matrix with microfluidic channels that regulate transport of
fluids and soluble factors (West et al., 2008). Further integration with bioanalytic
microsystems results in multifunctional platforms for basic biological insights into cells and
tissues, as well as for cell-based sensors with biochemical, biomedical and environmental
functions. Highly integrated microdevices show great promise for basic biomedical and
pharmaceutical research, and for drug discovery (Dittrich & Manz, 2006). Microfluidic lab

On-Chip Living-Cell Microarrays for Network Biology

611
Cell type
Batch/ micro-
fluidics
Array size
(wells, spots)
Characteristics References
Carcinoma cells Microfluidics 1
Cells immobilised with peptide
gel
Kim et al., 2007
Hepatocytes Batch microarray 512
Dynamic monitoring of
fluorescet probes in single cells
Roach et al.,
2009
Fibroblast Microfluidics 4
Ligand labeling and cell binding
analysis
Sui et al., 2007
Fibroblast 3T3 Microfluidics 96
Response of single cells to
different concentration of
signalling molecule (TNF)-
Tay et al., 2010
Fibroblast 3T3 Microfluidics 16 Perfusion culture for 3 days Kim et al., 2006
Fibroblast 3T3 Microfluidics 32
Quantitative interrogation of
signalling networks
Cheong et al.,
2009
H 35 cells Microfluidics 64/100
8x8 array with individually
addressable rows/10x10 array
Hung et al.,
2005; Lee et al.,
2006
Hela-NF Microfluidics 40
8x5 array: row with 5 wells is
individually addressed; GFP-
based gene expression
Thompson et al.,
2004
Hela-NF Microfluidics 256
16x16 array; GFP-based gene
expression
Wieder et al.,
2005
Human stem
cells
Batch microarray 1700
Interaction of biomaterials with
cells
Anderson et al.,
2004
Human stem
cells
Microfluidics 96
Transient stimulation schedules
on proliferation, differentiation
and motility
Gmez-Sjberg
et al., 2007
Human neural
SC
Microfluidics 1 Growth and differentiation
Chung et al.,
2005
mESC Microfluidics 16
Proliferation is flow rate
dependent, 4 days culture
Chin et al., 2004
mESC Batch microarray 280
Cells immobilised in alginate gel
spots
Fernandes et al.,
2010
Rat stem cells Batch microarray 10000
Dimensions of the wells are
tunable, diameter: 20 to >500m,
height: 10-500 m
Chin et al., 2004
Table 1. Examples of mammalian and stem cell microarrays/wells.
on a chip technologies have been used to track gene expression changes in individual cells,
enabling large populations of cells to be monitored, and allowing precise control of the cell
microenvironment (Breslauer et al., 2006; Charvin et al., 2009).
Conventional methods of fabricating microfluidic devices have centered on etching in glass
and silicon (Pisani & Tadigadapa, 2010). Polymers have assumed the leading role as substrate
materials for microfluidic devices in recent years (Becker & Grtner, 2008). They offer a broad
range of material parameters as well as material and surface chemical properties, which


612
enable microscopic design features that cannot be realised by any other class of materials.
Today, the most preferred material for biocompatible microfluidic devices is
poly(dimethylsiloxane) (PDMS) (Velve-Casquillas et al., 2010), which was introduced as soft
lithography by Whitesides (Anderson et al., 2000). PDMS is soft, transparant, permeable to
gasses, for most, impermeable to liquids, biocompatible, nontoxic, and has a low electrical
conductivity, making it a very suitable material for biological applications in microfluidic
devices. Fabrication of microfluidic devices in PDMS by soft lithography provides faster, less
expensive routes than these conventional methods to devices that handle aqueous solutions.
Soft lithography refers to a collection of techniques for creating microstructures and
nanostructures based on printing, moulding and embossing (Weibel et al., 2007). It is based
on rapid prototyping and replica molding. In rapid prototyping, a computer-aided design
program is used to create a design for channels, which are printed at high resolution onto
transparency film. The transparency film then serves as the photomask. The master molds are
generated by using the photomask in contact lithography to produce a positive relief of
photoresist. In replica molding, PDMS is poured over the master and heat cured to generate a
negative replica of the master. The PDMS is then removed from the mold and sealed against
a glass coverslip to form the device features and channels. Flows in microfluidic devices are
mainly pressure-driven by using syringe pump, rotary pump, or electro-osmotic flow.
Microfluidic devices are advantageous for cell assays for various reasons. The most obvious
one is the similarity in dimensions of cells and microchannels (10-100 m widths and
depths). Another important advantage is flow: fluid flow in these small channels is laminar.
Consequently, convection only exists in the direction of the applied flow, whereas in the
direction perpendicular to the applied flow, diffusion contributes to mass transport.
Although diffusion-based transport is slow across long distances, in microchannels diffusion
enables rapid reagent delivery. In addition, the combination of laminar flow and diffusion
makes the formation of highly resolved chemical gradients across small distances. This
feature is particular useful for cell assays as such gradients are common in living systems
(but difficult to implement in macroscale setups). Another advantage is the increased
surface-to-volume ratio, which facilitates favourable scaling of heat and mass transfer, as
well as favourable scaling of electrical and magnetic fields that are used in electromagnetic
cell analysis. Another consequence of the size regime lies in the concentration of analytes: as
cells in microchannels are confined in sub-microliter volumes, relevant analytes do not
become too dilute and can thus be more readily detected. A limitation of the high surface-to-
volume ratio of microchannels is the adsorption of molecules onto channel walls that are
generally hydrophobic. However, surfaces can be chemically treated to prevent adsorption
of biomolecules (Velve-Casquillas et al., 2010). Automated high-throughput experiments
may be performed in a large number of repeating functional microstructures fabricated on a
single chip. These microsystems can also monitor the time course of the release, which is
difficult to measure by conventional batch cell culture methods. Microfluidic devices can be
made transparant and the cells monitored in real time by imaging, using fluorescence
markers to probe cell functions and cell fate.
In a microfluidic device for cell-based assays, adequate culture conditions must be
maintained for the duration of the experiment, which can span several days. While being
cultured, cells must be continuously perfused with nutrients and oxygen; in addition,
constant temperature and pH must be maintained. In contrast to traditional batch cultures,
miniaturised perfusion systems provide precise control of medium composition, long-term
unattented cultures and tissue-like structuring of cultures (Heiskanen et al., 2010). Adherent


613
cells must be detached from culture flasks and seeded or spotted into a microfluidic device
while sufficient time has to be allowed to achieve proper cell attachment and reduction of
stress induced by the transfer. Mobile cells in suspension are easier to handle and require
less time to adapt to the new environment.
2.3 Single-cell analysis/monitoring in microfluidic devices
A fundamental goal of cell biology is identifying how cell behaviour arises from the dynamic
collection of environmental stimuli to which the cell is exposed (Lee & Di Carlo, 2009). From
a biosystems science and engineering perspective, there is great interest in how the cell
behaves as a system that processes time-dependent input signals into output behaviour(s).
Ideally, with knowledge of the history of the ensemble of environmental stimuli, one would
be able to predict the precise behaviour that a particular cell would exhibit under a given
stimulus. Unfortunately, cells under seemingly identical environmental conditions often
display a distribution of heterogeneous behaviour(s) (Lidstrom & Meldrum, 2003). This
appears to be partly due to probabilistic behaviour in the decision processes that connect
input and output (Raser & O'Shea, 2005; Mettetal et al., 2006). Underlying the links between
inputs and outputs are systems of interconnected molecular interactions (signalling
pathways). Signalling within one pathway as well as cross-signalling between pathways,
localisation of reactions and the sometimes small molecule numbers involved in signalling
contribute to stochastic behaviour in these systems (Raser & O'Shea, 2005; Kholodenko et al.,
2010), which in the case of stem cells may very well be an essential and necessary feature of
their biology and enables them to transit from one state to another. Because of the meanwhile
well-documented heterogeneity within such cell population, increased emphasis has been
put on analysing a large number of single cells and determining distributions of responses
(Cai et al., 2006; Mettetal et al., 2006; Yu et al., 2006). New tools, based on microfabrication and
microfluidic technologies, are now allowing improved dynamic control of environmental
variables for high-throughput single-cell analysis. These experimental technologies combined
with systems analysis of signalling pathways are expected to lead to an improved
quantitative description of single-cell function (Lee & Di Carlo, 2009).
Several single-cell analysis techniques have been developed, which may be classified in
terms of information content (number of elements capable of being studied simultaneously)
and throughput (number of cells studied in a give time). The simplest and most widely used
forms of single-cell analysis are fluorescence microscopy and flow cytometry. Automated
microscopy techniques, often termed high-content screening (HCS) or cellomics, recently
provided also quantitative insight into cellular behaviour and in most cases are applied to
observe the response of the cells, e.g. to drug candidate molecules.
The utility of single-cell measurements with high temporal resolution has been
demonstrated by bacterial studies, which used optical microscopy to observe Escherichia coli
over long time periods and reveal interesting temporal fluctuations and cell-to-cell
variability that would otherwise be masked by population-wide measurements (Pedraza &
van Oudenaarden, 2005). A microfluidic microchemostat has been constructed and used to
acquire single-cell fluorescence data from Saccharomyces cerevisiae over many cellular
generations (Charvin et al., 2009; Rowat et al., 2009). One way in which cells can rapidly
respond to environmental stimuli is to alter the localisation and abundance of proteins
(Charvin et al., 2009). In a microfluidic device, these aspects can be studied on the same cells
under various growth conditions or in response to environmental insults.


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2.4 Localisomics
Localisomics seeks to identify the subcellular location of all proteins in the cell, which can
provide key insights into the cellular function of the individual proteins a well as their
probable interacting partners (Joyce & Palsson, 2006). Protein localisation has to be
described in intracellular compartments, e.g. the nucleus or cytoplasm, and also in
organelles, as specialisation of cellular organelles defines the functional roles of proteins
(Souchennytskyi, 2005). The most informative is data about protein localisation and its
dynamics in a single, living cell.
Mostly fluorescence microscopy techniques have been used to monitor green fluorescent
protein (GFP)-tagged- or yellow fluorescence protein (YFP)-tagged proteins in E. coli
(Taniguchi et al., 2010), S. cerevisiae (Huh et al., 2003) and human cells (Shariff et al., 2010).
Visual interpretation of the fluorescent images, and more recently, automated image
analysis, have been used to extract dynamic protein localisation data (Schubert et al., 2006;
Conrad et al., 2011). Images from many studies are publicly available (Table 2).

Species (cell type)
Number of
proteins
Tagging method Website Reference
Human (U-2 OS,
A-431, U-251 MG)
> 6000
Immuno-fluorescence,
immunochemistry
www.proteinatlas.org Berglund et al., 2008
Mouse (3T3) >100 Internal GFP fusion cdtag.bio.cmu.edu Jarvik et al., 2002
Human (HeLa)
Monkey (Vero)
>1000
cDNA terminal GFP
fusion
gfp-cdna.embl.de Liebel et al., 2004
Human (HeLa)
Mouse (3T3)
>100
Immunofluorescence
and genomic internal
GFP fusion
murphylab.web.cmu.edu Huang et al., 2002
Human (H1299
carcinoma)

> 2000 YFP CD tagging
www.dynamicproteomics
.net
Frenkel-Morgenstern
et al., 2010
Yeast > 4000
cDNA C-terminal
GFP fusion
yeastgfp.yeastgenome.org Huh et al., 2003
Human (brain)
Various
Various Various ccdb.ucsd.edu Martone et al., 2008
Table 2. Publicly available microscopy images concerning protein localisation in cells
(adapted from Newberg et al., 2009).
3. Computational methods for quantitative image analysis
Quantitative information from live cell microscopy can be obtained. To reach this goal,
image analysis methods have to be used. These methods can provide quantified geometric,
intensity, and motion properties, and these quantitative parameters can be used as input
parameters for predictive systems biology models (Pepperkok & Ellenberg, 2006; Megason
& Fraser, 2007; Bakal et al., 2007; Verveer & Bastiaens, 2008).
Advances in imaging technology provide a huge amount of digital image data. A manual
analysis is hardly possible. Additionally, 3D images over time are difficult to interpret
manually and the result suffers from subjectivity. Therefore, computer-based image analysis
is required to cope with the enormous amount of image data and to extract reproducible as


615
well as quantitative information (Peng, 2008; Zhou & Wong, 2008; Hamilton, 2009; Swedlow
et al., 2009; Rohr et al., 2010).
Automatic analysis of multidimensional live cell microscopy images requires different
computational methods. A general workflow for quantitative analysis of live cell
microscopy images is composed of the following steps: (i) preprocessing, (ii) segmentation,
(iii) registration, (iv) tracking and (v) classification (Rohr et al., 2010).
3.1 Preprocessing
The goal of image preprocessing is to improve the quality of raw images prior to image
segmentation and feature extraction. Applications include denoising for reducing the image
noise, elimination of artifacts, intensity normalisation, contrast enhancement, and
deconvolution for reducing the image blur introduced by the imaging process. Denoising
methods use either linear or nonlinear filters to reduce noise in images and improve the
signal-to noise ratio. For denoising images, a Gaussian filter is often applied (Rohr et al.,
2010). Filters that are not based on convolution are called nonlinear filters. A nonlinear filter
that is often used to remove the pepper-noise generated by CCD detectors in optical
fluorescent microscopy is the median filter (Zhou & Wong, 2008). This median filter can
preserve high frequency information describing cell edges in high content microscopy
images.
Deconvolution methods to reduce the image blur are relevant for both wide-field and
confocal light microscopes (Cannell et al., 2006). It is often assumed that the blurring of an
image is caused by a linear process and thus can be presented by convolution with a point
spread function (PSF). The aim of deconvolution is to reconstruct the original (true) image
by reversing the effect of convolution and thus improving the resolution and contrast of the
image (Rohr et al., 2010). Examples of such approaches are the inverse filter, the Wiener
filter, and the constrained least-squares filter.
3.2 Segmentation
Image segmentation is one of the most basic processing steps in many bioimage informatics
applications. The goal is to segment out meaningful objects of interest in the respective
image. In the case of microscopy images, one main task is to identify cells and to distinguish
them from the background. Another task is to detect and localize particles in the image.
Because particles are much smaller than cells and corresponded to spot-like image
structures, different segmentation methods are required for cells and particles (Rohr et al.,
2010). Segmentation is a prerequisite for quantifying geometric properties of objects as well
as for quantifying the corresponding signal intensities. Additionally, segmentation is often
the basis for subsequent image analysis steps, i.e. for tracking.
3.2.1 Cell segmentation
Cell segmentation can be categorised into two classes, i.e. nucleic segmentation and
cytoplasm (or whole cell) segmentation. In recent years, there has been significant effort
towards the development of automated methods for cell nuclei image and 3D cell
segmentation have been developed (Ortiz de Solorzano et al., 1999; Sarti et al., 2000; De
Solorzano et al., 2001; Umesh Adiga & Chaudhuri, 2001; Malpica et al., 1997; Belien et al.,
2002; Whlby et al., 2004; Lin et al., 2005; Lindblad et al., 2004; Dufour et al., 2005; Li et al.,
2007, 2008; Dorn et al., 2008; Ko et al., 2009). The main methods for cell segmentation can be


616
classified as: threshold-based segmentation, edge-based segmentation, region-based
segmentation, and deformable models (reviewed in Rohr et al., 2010).
3.2.2 Particle localisation
Often it is assumed that the intensities representing a fluorescently labelled particle
resemble a 2D Gaussian function in which the peak intensity value of the particle differs
significantly from that of the local background. A bottom-up or a top-down strategy is used
to address the problem of particle localisation.
Bottom-up localisation schemes for fluorescent particles typically comprise three
consecutive steps: image preprocessing, particle detection, and particle localisation (Rohr et
al., 2010). A common technique is to apply a threshold on the intensities of a (preprocessed)
image to determine image regions that correspond to particles (Ponti et al., 2003; Sbalzarini
& Koumoutakos, 2005). Automatic schemes for determining an optimal threshold is
required since manual determination is often impractical and can give inconsistent results.
Top-down approaches use model-driven strategies in which hypotheses regarding the
possible configuration of the models are tested against the information found in the images.
A 2D Gaussian function is typically used as a model for the shape and appearance of
fluorescently labelled particles (Godinez et al., 2007; Cortes & Amit, 2008).
3.3 Registration
The task of finding an optimal geometric transformation between corresponding image data
is known as registration. Bioimage registration is essential in many applications that need to
compare multiple image subjects of different conditions. Registration approaches can be
classified based on the type of transformation model and image information used (Rohr et
al., 2010). The transformation model defines the degrees of freedom for geometrically
matching two images, and a main distinction is made between rigid, affine, and nonrigid
schemes.
Many of the 2D and 3D image registration methods proposed for medical image analysis,
such as the mutual information registration (Viola & Wells, 1997), spline-based elastic
registration (Rohr et al., 2003), invariant moment feature-based registration (Shen &
Davatzikos, 2002), congealing registration (Miller, 2006; Zollei et al., 2005), etc., can be
extended to align the molecular and cellular images (Peng, 2008). Nonrigid or elastic
registration approaches are required to cope with the shape changes of live cells (Rohr et al.,
2010). An intensity-based nonrigid registration approach for cell microscopy images, which
relies on an optic flow scheme and uses segmented images, has been developed recently
(Yang et al., 2008). An intensity-based approach has been used to register segmented 2D
static images of different cell nuclei (Rohde et al., 2008), and a biomechanical model has been
used to register 3D segmented images of cell nuclei (Gladilin et al., 2008). An intensity-based
nonrigid registration approach that directly analyses the intensity information without
requiring a segmentation step has been developed (Kim et al., 2007). This approach relies on
optic flow estimation and has been applied to register 2D and 3D time-lapse images of live
cells for accurate analysis of protein particle movement.
3.4 Tracking and motion analysis
Dynamic cell population studies are becoming more and more important in understanding
pathways and networks (Glory and Murphy, 2007). Live cell fluorescent video microscopy


617
offers a wealth of information on the dynamic organisation of proteins and subcellular
structures that is unavailable in static 2D and 3D imaging. With the addition of time,
organelle dynamics as proteins are recruited, transported and expelled can be viewed in
detail and the passage through a cell of proteins and the structures that they interact with
can be readily observed (Hamilton, 2009). Additionally, the addition of temporal parameters
such as the change of size and size of nuclei and the duration between the different stages
are important indicators of the cell division cycle (Zhou and Wong, 2006). There is also
extensive work on analysing the behaviour of specific labelled proteins by tracking
individual objects in time series images (Meijering et al., 2006).
Tracking denotes the repeated localisation of objects in successive images. The aim is to
establish temporal correspondences between objects to analyse object motion (Rohr et al.,
2010). Although finding correspondences is largely simplified when there is only one object in
the images, this task is generally quite challenging when there are several or a large number of
moving objects. Therefore, sophisticated multiple target tracking methods are required.
Object tracking from fluorescent video microscopy present many challenges (Hamilton,
2009). Objects viewed may join, split, disappear, change direction or substantially change
their morphology, and there are technical challenges such as photobleaching and
compromises between spatial and temporal resolution. Tracking algorithms developed in
other research areas and adapted to fluorescent video microscopy tend to perform poorly
and considerable research has gone into designing algorithms specific to fluorescent
imaging (reviewed in Kalaidzidis, 2009).
3.5 Classification
A last step in image analysis is to distinguish objects into different classes. Automatic
classification methods can be divided into supervised and unsupervised learning methods
(Glory & Murphy, 2007). Supervised learning methods allow classification into predefined
classes and require training of the classifier with a set of annotated examples. In
unsupervised learning methods, the classes do not need to be known in advance.
Supervised learning methods are used for cell microscopy since the classes are known in
advance. Common used classifiers are artificial neural networks (Boland & Murphy, 2001),
k-nearest-neighbour classifiers (Chen et al., 2006), and support vector machines (Conrad et
al., 2004; Huang & Murphy, 2004; Harder et al., 2008).
4. Biological network analysis
4.1 Network modelling
Network modelling is a key step for processing dynamic proteomics data, because a
network model provides: (i) a means of understanding how detected proteins are associated
with underlying network operations, and (ii) a platform into which other useful
information, (such as protein abundances and localisation) can be integrated.
A cell is an enormous complex entity made up by myriad interacting molecular components
that perform the biochemical reactions that maintain life. A cell can be described through
the set of interconnections between its component molecules according to the network
hypothesis (De Los Rios & Vendruscolo, 2010). The central dogma in molecular biology
describes the way in which a cell processes the information required to produce the
molecules necessary to maintain life and reproduce (Crick, 1970). In order to obtain a more
complete description of the functioning of a cell, a deeper understanding of the manner in


618
which the sets of interconnections between these molecules are defining the identity of the
cell itself is needed (De Los Rios & Vendruscolo, 2010). Therefore, it is important to
investigate whether the genetic makeup of an organism does not only specify the rules for
generating proteins, but also the way in which these proteins interact among themselves
and with the other molecules in a cell. Networks provide a way to organise and regulate
efficiently complex systems. In an effective network different parts are linked by reducing at
a minimum the number of interconnections. A network is also a powerful method to
represent the data in one object and to enable the quantitative assessment of the fragility or
robustness of the system. The biological molecules in a cellular system are individual
molecules, which affect each other by pairwise interactions (Chen et al., 2009). A cascade of
those pairwise interactions forms a local structure (i.e. a linear pathway or a subnetwork),
which transforms local perturbations into a functional response. All linear pathways or
subnetworks are assembled into a global biomolecular network, which eventually generates
global behaviours and holds responsability for complicated life in a living organism.
Gene products, such as mRNA and proteins, are produced through the transcription and
translation processes. Gene, mRNA, and protein are known as biological molecules or basic
components (Chen et al., 2009). The complicated relations and interactions between these
components are responsible for diverse cellular functions. Transcription factors (TFs) are
DNA-binding proteins that can activate or inhibit the transcription of genes to synthesise
mRNAs by regulating the activities of genes. Since these TFs themselves are products of
genes, the final effect is that genes regulate each other's expression as part of a transcription
(or translational) regulatory network (TRN) or gene regulatory network (GRN). At the
proteome level, proteins participate in diverse posttranslational modifications of other
proteins or form protein complexes and pathways together with other proteins. Such local
associations between proteins molecules are called protein-protein interactions (PPIs), which
form a protein interaction network. The biochemical reactions in cellular metabolism can
likewise be integrated into a metabolic network whose fluxes are regulated by enzymes that
catalyse the reactions. In many cases, these interactions at different levels are integrated into
a signaling network.
Multiple proteins in a cell are in dynamic interaction with each other, and these interactions
provide functioning and behaviour of living cells (Terentiev et al., 2009). Reversible protein-
protein interactions are among other dynamic processes that proceed in a cell and contribute
to cell functioning. The whole set of protein-protein interactions of a given organism is
referred to as the "interactome". Structural organisation of interactomes and the total
number of interactions in them are important factors that determine complexity of biological
systems. The number of copies of a certain protein per cell can vary from several tens to
millions (Ghaemmaghami et al., 2003). Interactomes even of simple organisms can be formed
by a rather large number of interactions. The determination of physically interacting protein
pairs makes it possible to design interactome maps as graphs consisting of nodes, in which a
particular protein is located, and of links between them that indicate paired interactions. The
interactome maps are considered as keys to obtain knowledge on protein functioning (Rual
et al., 2005).
4.2 Integration of biological networks
4.2.1 Network visualisation and analysis
Many tools exist for visually exploring networks and network analysis, including examples
such as Cytoscape (Shannon et al., 2003), VisANT (Hu et al., 2009), Osprey (Breitkreutz et al.,


619
2003), CellDesigner (Kitano et al., 2005), BioLayout (Goldovsky et al., 2005), GenMAPP
(Dahlquist et al., 2002), PIANA (Aragues et al., 2006), ProViz (Iragne et al., 2005), and Patika
(Demir et al., 2002). These systems play a key role in the development of integrative biology,
systems biology and integrative bioinformatics. The trend in the development of these tools
is to go beyond static representations of cellular states, towards a more dynamic model of
cellular processes through the incorporation of gene expression data, subcellular localisation
information and time-dependent behaviour (Suderman & Hallett, 2007).
Cytoscape is an open source software project for integrating biomolecular interaction
networks with high-throughput expression data and other molecular states into a unified
conceptual framework (Shannon et al., 2003). In Cytoscape, nodes representing biological
entities, such as proteins or genes, are connected with edges representing pairwise
interactions, such as experimentally determined proteinprotein interactions. Nodes and
edges can have associated data attributes describing properties of the protein or interaction.
A key feature of Cytoscape is its ability to set visual aspects of nodes and edges, such as
shape, color and size, based on attribute values. This data-to-visual attribute mapping
allows biologists to synoptically view multiple types of data in a network context.
Additionally, Cytoscape allows users to extend its functionality by creating or downloading
additional software modules known as plugins.
VisANT is a web-based software framework for visualising and analysing many types of
networks of biological interactions and associations (Hu et al., 2005). Given user-defined sets
of interactions or groupings between genes or proteins, VisANT provides: (i) a visual
interface for combining and annotating network data, (ii) supporting function and
annotation data for different genomes from the Gene Ontology and KEGG databases, and
(iii) the statistical and analytical tools needed for extracting topological properties of the
user-defined networks. The new VisANT (v3.5) functions can be classified into three
categories (Hu et al., 2009). (i) Visualisation: a new tree-based browser allows visualisation
of GO hierarchies. GO terms can be easily dropped into the network to group genes
annotated under the term, thereby integrating the hierarchical ontology with the network.
This facilitates multi-scale visualisation and analysis. (ii) Flexible annotation schema: in
addition to conventional methods for annotating network nodes with the most specific
functional descriptions available; VisANT also provides functions to annotate genes at any
customized level of abstraction. (iii) Finding over-represented GO terms and expression-
enriched GO modules: two new algorithms have been implemented as VisANT plugins.
One detects over-represented GO annotations in any given sub-network and the other finds
the GO categories that are enriched in a specified phenotype or perturbed dataset. Both
algorithms take account of network topology (i.e. correlations between genes based on
various sources of evidence).
Osprey is a Java-based network visualisation and analysis tool for protein-protein and
genetic interaction networks (Breitkreutz et al., 2003). Osprey builds data-rich graphical
representations that are color-coded for gene function and experimental interaction data.
Mouse-over functions allow rapid elaboration and organisation of network diagrams in a
spoke model format. User-defined large-scale datasets can be readily combined with Osprey
for comparison of different methods.
GenMAPP is a free computer application designed to visualise gene expression and other
genomic data on maps representing biological pathways and groupings of genes (Dahlquist
et al., 2002). Integrated with GenMAPP are programs to perform a global analysis of gene
expression or genomic data in the context of hundreds of pathway MAPPs and thousands of


620
Gene Ontology Terms (MAPPFinder), import lists of genes/proteins to build new MAPPs
(MAPPBuilder), and export archives of MAPPs and expression/genomic data to the web.
The main features underlying GenMAPP are: (i) draw pathways with easy to use graphics
tools, (ii) color genes on MAPP files based on user-imported genomic data, (iii) query data
against MAPPs and the GeneOntology.
CellDesigner is a structured diagram editor for drawing gene-regulatory and biochemical
networks (Kitano et al., 2005). Networks are drawn based on the process diagram, with
graphical notation system proposed by Kitano, and are stored using the Systems Biology
Markup Language (SBML), a standard for representing models of biochemical and gene-
regulatory networks. Networks are able to link with simulation and other analysis packages
through Systems Biology Workbench (SBW). CellDesigner supports simulation and
parameter scan by an integration with SBML ODE Solver and Copasi. By using
CellDesigner, you can browse and modify existing SBML models with references to existing
databases, simulate and view the dynamics through an intuitive graphical interface.
BioLayout uses a general approach for the representation and analysis of networks of
variable type, size and complexity (Goldovsky et al., 2005). The application is based on the
original BioLayout program (C-language implementation of the Fruchterman-Rheingold
layout algorithm), entirely re-written in Java to guarantee portability across platforms.
BioLayout(Java) provides broader functionality, various analysis techniques, extensions for
better visualisation and a new user interface.
PIANA (Protein Interactions And Network Analysis) facilitates working with protein
interaction networks by (i) integrating data from multiple sources, (ii) providing a library
that handles graph-related tasks and (iii) automating the analysis of protein-protein
interaction networks (Aragues et al., 2006). PIANA can also be used as a stand-alone
application to create protein interaction networks and perform tasks such as predicting
protein interactions and helping to identify spots in a 2D electrophoresis gel.
ProViz is a tool for the visualisation of protein-protein interaction networks, developed by
the IntAct European project (Iragne et al., 2005). It provides facilities for navigating in large
graphs and exploring biologically relevant features, and adopts emerging standards such as
GO and PSI-MI.
Patika (Pathway Analysis Tool for Integration and Knowledge Acquisition) is based on an
ontology for a comprehensive representation of cellular events (Demir et al., 2002). The
ontology enables integration of fragmented or incomplete pathway information and
supports manipulation and incorporation of the stored data, as well as multiple levels of
abstraction. Patika is composed of a server-side, scalable, object-oriented database and
client-side editors to provide an integrated, multi-user environment for visualising and
manipulating network of cellular events. This tool features automated pathway layout,
functional computation support, advanced querying and a user-friendly graphical interface.
4.2.2 Subcellular localisation
Interesting tools that take into account the subcellular localisation are (Suderman & Hallett,
2007): the Cytoscape plugin Cerebral (Barsky et al., 2007), Patika (Demir et al., 2002; see
4.2.1), and Cell Illustrator (Nagasaki et al., 2010). Cerebral (Cell Region-Based Rendering and
Layout) is an open-source Java plugin for the Cytoscape biomolecular interaction viewer.
Given an interaction network and subcellular annotation, Cerebral automatically generates a
view of the network in the style of traditional pathway diagrams, providing an intuitive
interface for the exploration of a biological pathways or system. The molecules are separated


621
into layers according to their subcellular localisation. Potential products or outcomes of the
pathway can be shown at the bottom of the view, clustered according to any molecular
attribute data-protein function, for example. Celebral scales well to networks containing
thousands of nodes.
Patika partitions the drawing space into regions corresponding to the subcellular
localisations and then search for layouts where nodes are forcibly constrained to their
respective locations (Demir et al., 2002). It makes use of a modified force-directed algorithm
to achieve this.
Cell Illustrator is a software platform for systems biology that uses the concept of Petri net
for modeling and simulating biopathways (Nagasaki et al., 2010). It is intended for biological
scientists working at bench. The recent version of Cell Illustrator 4.0 uses Java Web Start
technology and is enhanced with new capabilities, including: automatic graph grid layout
algorithms using ontology information; tools using Cell System Markup Language (CSML)
3.0 and Cell System Ontology 3.0; parameter search module; high-performance simulation
module; CSML database management system; conversion from CSML model to
programming languages (FORTRAN, C, C++, Java, Python and Perl); import from SBML,
CellML, and BioPAX; and, export to SVG and HTML. Cell Illustrator employs an extension
of hybrid Petri net in an object-oriented style so that biopathway models can include objects
such as DNA sequence, molecular density, 3D localisation information, transcription with
frame-shift, translation with codon table, as well as biochemical reactions.
4.2.3 Network integration for cellular microarray data using Cytoscape
Data from cellular microarray experiments include a list of differentially expressed proteins,
i.e. changed fluorescence intensity (protein abundance), as a function of time and
localisation in the cell. Integration of these data with other available biological network data
for a specific organism can be performed using the above listed software platforms (see
4.2.1), e.g. by using Cytoscape supplemented with the available plugins.
The cellular microarray data can be mapped to the protein interactome. Network data
related to these proteins can be imported into Cytoscape using three options: querying
interaction databases using cPath (Cerami et al., 2006), building an association network
through text mining using Agilent Literature Search plugin (Vailaya et al., 2005), and
loading own network data from a text file. Additionally, pathways from repositories, such
as KEGG (Wixon & Kell, 2000), Reactome (Joshi-Tope et al., 2005) via the PSI-MI, BioPAX, or
SBML data exchange formats (Strmbck et al., 2006), can be imported.
Networks can be analysed further using topologic information, and using combined
information of various types, such as GO annotations and known pathways. Network
modules enriched by GO terms and pathways (functional enrichment) can be identified.
Therefore, the Cytoscape plugins BiNGO (Maere et al., 2005) and DAVID (Dennis et al., 2003;
Huang da et al., 2009) can be employed. GO Biologic Process (GOBP) trees with nodes
corresponding to GOBP terms is generated using the BiNGO plugin. GOBP terms that differ
in terms of their degrees of enrichment can be identified, as can sets of network nodal
proteins belonging to such GOBP terms. Pathways enrichment analysis can be performed
for network nodal proteins using DAVID.
Network structures and active subnetworks can be explored using the Cytoscape plugins
MCODE (Bader et al., 2003) and jActiveModules (Ideker et al., 2002). The MCODE-plugin can
be used to generate network clusters within which proteins are densely connected, whereas
proteins across different network clusters loosely interact. Both the core network modules and
their dynamic relationships can be identified by integrating time-dependent protein


622
abundance information. In addition, active networks can be identified among network
modules using jActiveModules, which select networks with high collective abundances.

Fig. 1. Work scheme for on-chip cellular microarray screening and biological network
analysis.
5. Summary
In this chapter, on-chip living-cell microarrays to study network biology is reviewed. A
general work scheme is shown in Figure 1. Microfluidic technology holds great promise for
the creation of advanced cell culture models. It can be used in combination with time-
lapse fluorescent microscopy, and image analysis and data mining to observe multiple
cellular components over time in individual cells, i.e. dynamics of a FP-tagged protein.
Integration of dynamic localisomics data with other avialable biological network data allows
performing a quantitative system-wide analysis for a particular cell.
Cell assays in microfluidic chips that have been used for cellular microarrays are discussed
in detail in this chapter. Next, image analysis algorithms to extract dynamic proteomics data
from cellular microarray experiments are reviewed. In the last part, the integration of the
cellular microarray data into a network model, as well as network analysis options are
discussed.
6. Acknowledgment
R. Willaert is supported by the Belgian Federal Science Policy Office and European Space
Agency (ESA) PRODEX program, the Institute for the Promotion of Innovation by Science
and Technology in Flanders (IWT) and the Research Council of the VUB.


623
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28
Novel Machine
Learning Techniques for
Micro-Array Data Classification
Neamat El Gayar, Eman Ahmed and Iman El Azab
Faculty of Computers and Information,
Cairo University,
Egypt
1. Introduction
Machine learning, data mining and pattern recognition have been quite often used in
various contexts of medical and bioinformatics applications. Currently computational
methods and tools available for that purpose are quite abundant. The main aim of this
chapter is to outline to the practitioners the basic concepts of the fields focusing on essential
machine learning tools and highlighting their best practices to be successfully used in the
medical domain. We present a case study for DNA microarray classification using ensemble
methods and feature subset selection techniques.
The background section will begin by introducing the reader to the fields of pattern
recognition, machine learning and data mining. It will then focus on some of the most
important concepts related to machine learning.
In particular in section 2 we review the most popular machine learning models for
classification used in the context of the medical domain. We then describe one of the most
powerful and widely used classifiers for high dimensional feature spaces; the support vector
machines (SVM). We cover the area of classifier evaluation and comparison to provide
practitioners with essential understanding of how to test, validate and select the appropriate
models for their applications. Finally, we summarize the main advances in the field of
ensemble learning, feature subset selection and feature subset ensembles.
Section 3 presents a review of using machine learning in various fields of bioinformatics.
In section 4, a recent case study on DNA microarray data that uses an ensemble of SVMs
coupled with feature subset selection methods is presented. We show how the proposed
model can alleviate the curse of dimensionality associated with expression-based
classification of DNA data in order to achieve stable and reliable results.
Section 5 describes the data used and the experiments conducted, while section 6 presents
results and a comparative analysis for the proposed models.
Finally in section 7 we summarize the main contributions of this chapter and review the
main guidelines to effectively use machine learning tools. We end this section by
highlighting a set of challenges that need to be addressed and propose some future research
directions in the field.

2. Background
2.1 Pattern recognition, machine learning and data mining
Pattern recognition can be defined as the categorization of the input data into identifiable
classes via the extraction of significant features or attributes of the data from a background
of irrelevant detail (Duda et al, 2000). The task of pattern recognition is also viewed as the
transformation from the measurement space to the feature space and finally to a decision
space.
Machine learning techniques aim at producing a system that can learn and adapt from the
environment and hence exhibits a kind of intelligence essential for applications that lack
known solutions (Alpydin, 2004). Machine learning models very often attempt to optimize a
criterion function through exploiting information from training examples.
Data mining, on the other hand, can be thought of as a collection of statistical, machine
learning, pattern recognition and artificial intelligence tools that help uncover and extract
hidden knowledge from data. Particularly in the medical domain data mining refers often to
techniques and methods that analyze large amounts of data. These techniques include among
many others classification, clustering, association rule mining and regression or prediction.
Cluster analysis usually addresses segmentation problems. The objective of this analysis is to
separate data with similar characteristics from the dissimilar ones. Cluster analysis is
frequently the first required task of the mining process. Cluster analysis can also be used for
outlier detection to identify samples with peculiar behavior. Among the most simple and
efficient clustering techniques are K-means, fuzzy K-means, Self Organizing maps; in
addition to more advanced clustering methods like evolving clustering techniques and
distributed clustering.
The purpose of association rule mining, on the other hand, is to search for the most significant
relationship across large number of variables or attributes. Sometimes, association is viewed as
one type of dependencies where affinities of data items are described (e.g., describing data
items or events that frequently occur together or in sequence). Some techniques for association
analysis are nonlinear regression, rule induction, Apriori algorithm and Bayesian networks.
Time Series prediction is also an important aspect in data mining whereby the temporal
structure and ordering of the data is utilized to estimate some future value based on current
and past data samples. Time-series prediction encompasses a wide variety of applications.
As mentioned earlier, the purpose of this chapter is to provide a broad introduction to the
fundamentals of machine learning suitable for bioinformatics. The rest of the chapter will
mainly focus on the classification problem.
2.2 Machine learning models for classification
Classification is usually referred to as the process of devising models that can predict
categorical (discrete, unordered) class labels. Often machine learning models are used for
these purposes that learn the class functions using a set of given training examples.
Popular machine learning classification models are decision tree classifiers, Bayesian
classifiers, Bayesian belief networks, rule based classifiers and Backpropagation- Multi layer
neural network (Hand et. al, 2001). More recent approaches to classification include support
vector machines and ensemble methods. In addition, other approaches are frequently
encountered in the literature like k-nearest-neighbor classifiers, case-based reasoning,
genetic algorithms, rough sets and fuzzy logic techniques.
According to a recent ranking (KDnuggets : Polls, 2006) common classification models used
in the data mining community are decision trees, decision rules, logistic regression, artificial

Novel Machine Learning Techniques for Micro-Array Data Classification 633
neural networks, support vector machines, the nave Bayesian classifier and Bayesian
networks.
It is worth mentioning at this point that particularly in medical applications sometimes
models are preferred that are more interpretable. Such models posses some characteristics
like being able to make knowledge discovered from data explicit and communicable to
domain experts, the provision of an explanation when deploying and using the
knowledge with new cases, in addition to the ability to encode and use the domain
knowledge in the data analysis process (Bellazi & Zupan 2008). Decision trees and
Bayesian networks are among the models that are easily explainable. Decision Trees are
sometimes preferred over more accurate classifier because of their descriptive power; i.e.
the ability interpret classification rule produced by the model. This is particularly
important for safety critically medical applications where results are required to be
understood by domain experts.
Moreover, the fact that medical data can often be imperfect is complemented in practice by
exploiting domain knowledge. Building classification models using background knowledge
is very useful in order to take into account information which is already known and should
not be rediscovered from data. Background knowledge can be expressed using different
models like Bayesian models, decision rules and fuzzy rules.
From another perspective, in the bioinformatics applications and in particular for the DNA
microarray data classification; more powerful tools are needed to deal with the challenges
posed by the low sample size, high dimensionality, noise and large biological variability
present in the data.
We therefore devote the next subsections for reviewing Support Vector Machines (SVMs),
ensembles methods and feature subset selection techniques. These techniques are known to
be robust tools for classification in noisy, high-dimensional and complex domains.
2.3 Support vector machines
This section is devoted to review one of the most powerful and widely used classifiers for
high dimensional feature spaces; the support vector machines (SVM).
SVMs are binary classifiers that aim to produce an optimal classifier that lies in midway
between the nearest data points of the 2 classes of the problem at hand.
In case of linearly separable problem, SVMs discriminate between two classes by fitting an
optimal separating hyper-plane in the midway between the closest training samples of the
opposite classes in a multi-dimensional feature space. This is done by maximizing the
margin which is the distance between the closest training samples and the classifier.
Given Z a training dataset with N samples in d-dimensional feature space R
d
.
Each x
i
has class y
i
= 1.
The objective is to find the linear hyper-plane represented by:
f(x) wx b = + (1)
Where w is the weight vector and b is the bias that maximizes the margin under the
constraint of correct classification. It was found that minimizing w maximizes the margin.
This forms the following optimization problem:

2
N
w
min C
i
2
i 1

+

=

(2)

With C as regularization parameter and as slack variables.
In case of non-linearly separable classes, the input samples are mapped to a high dimensional
feature space using a kernel function. Thanks to kernel trick, it is possible to work within the
newly transformed feature space without having to map every sample explicitly.
The training of the SVM requires getting optimal parameter values for the regularization
parameter C.
The final SVM function for non-linearly separable case is represented by:

N
f(x) y k x, x b
i i i
i 1
= +
=
(3)
Where
i
are Lagrange multipliers.
Further detailed explanation can be found in (Abe, 2005).
2.4 Ensemble learning
Ensemble classifiers - also called Multiple Classifier Systems (MCS) - are based on the
design of several classifiers separately then joining the final classification decision. MCS are
a preferred solution to recognition problems because it allows simultaneous use of different
feature descriptors of many types, corresponding measures of similarity and many
classification procedures. Examples of these techniques include bagging, boosting, and
mixtures of experts and others. Refer to (Roli & Giacinto 2002) (Kuncheva, 2004) (MCS
series) for a good review on methods and research in that area.
Perhaps the most obvious motivation for classifier ensembles is the possibility to boost the
classification accuracy by combining classifiers that make different errors or by combining
local experts. The fact that the best individual classifier for the classification task at hand is
very difficult to identify unless deep prior knowledge is available is also a motivation for
using multiple classifiers. Another reason is when the features of a sample may be presented
in very diverse forms, making it impossible to use them as input for one single classifier.
Another rationale is the desire to boost efficiency by using simple and cheap classifiers that
operate only on a small set of features. These are all cases that can be found in medical and
bioinformatics data.
Classifier combination can fall under one of the following taxonomies according to the type
of outputs produced by the classifiers (Kittler et al. 1998). Classifier outputs can be crisp
outputs (also called abstract level), ranked list of data classes or measurement level outputs.
For abstract level, a classifier outputs a unique label for every pattern to be classified. The
combination of such classifiers is usually done by voting strategies, such as majority vote,
weighted majority vote or by trained fusion rules such as Behavioural Knowledge Space
(Kuncheva, 2004).
For rank level classifiers, the output is a ranked list of labels for every pattern. Borda Count
is the common technique to combine these rankings. The rankings from all classifiers are
combined by ranking functions assigning votes to the classes based on their positions in the
classifiers' rankings. The final decision is taken as the minimum of the sum of these
rankings. Finally, at the measurement level, the classifier output represents the degree of
belongingness in each class. For this type of output various combination rules can be
applied like product, sum, mean, etc. These combination rules are derived mainly from
Bayesian decision rule. Non-Bayesian combinations can also be applied such that a
weighted linear combination of classifiers is learnt using optimization techniques.

In our model described in section 4, we present a combiner based on a SVM trainable
classifier that works on measurement level outputs of the base classifiers.
2.5 Feature subset selection and feature subset ensembles
A common way to build base classifiers for further combination is by randomly selecting
different subsets of features and training classifiers on those subsets. Feature subset selection
should enforce diversity among classifiers created and hence lead to more robust ensembles.
In applications that are characterized by having a huge number of features, feature subset
ensembles can be used in order to make use of all the features.
The way this method works is by sub-sampling the features such that the base classifiers in
the ensemble can be built on different subsets of features, either disjoint or overlapping. So
instead of over-whelming a single classifier with all the features, individual classifiers can be
built on groups of feature then their decisions are combined to get the final decision.
The choice of features for each subset depends on the problem at hand. The features may be
naturally grouped forming the feature subsets. They can also be selected by any available
feature selection method. The random subspace methods (Ho, 1998) work well when there is
redundant information dispersed across all the features (Kuncheva, 2004).
Also, various heuristic search techniques such as genetic algorithms, tabu search and
simulated annealing are used for feature subset selection. The feature subsets can be selected
one at a time or all at the same time in one run of the algorithm by optimizing some
ensemble performance criterion function (Kuncheva, 2004).
Random selection is the intuitive way for selecting samples and is the simplest method
available. It assumes a uniform distribution for all the samples.
There are two types of random selection: Random selection without replacement in which the
samples are randomly selected then removed so that they cannot be chosen again and
Random selection with replacement where the samples are randomly selected then placed back
so that they can be chosen again.
In our case study presented later, we use Random selection without replacement. We also
propose a feature subset selection method based on the K-means clustering algorithm. K-
means is a typical partition-based clustering method. Given a pre-specified number K, the
algorithm iteratively partitions the data set till it gets K disjoint subsets. In these iterations,
K-means tries to minimize the sum of the squared distances of the samples from their cluster
centres. It is a simple and fast algorithm. In our proposed approach the genes are the objects
of interest to be clustered and they are characterized by their expression values among the
samples in the microarray dataset.
2.6 Classifier testing and evaluation
As follows we present main concepts for classifier evaluation and comparison. We start by
reviewing cross validation and then discuss main performance measures that can be used to
evaluate classification results.
2.6.1 Cross validation
A classifier usually learns from the available data. The problem is that the resulting classifier
may fit on the training data, but might fail to predict unseen data.
Cross validation is a technique for assessing the generalization performance of a given
classifier. It can be used for estimating the performance of a given classifier as well as for
tuning the model parameters.

Methods of cross validation include Re-substitution Validation, Hold-Out Validation, K-Fold
Cross Validation, and Leave-One-Out Cross Validation as will be described next.
In Re-substitution Validation all the available dataset is used for training the classifier. Then, it
is tested on the same dataset. This makes it liable to overfitting. Thus, the classifier might
perform well on the available data yet poorly on future unseen test data. However in the
Hold-Out Validation the available dataset is split into 2 sets: one for training and the other for
testing the model, such that the model can be tested on unseen data. For this approach the
results are highly dependent on the choice for the training / test split. The instances in the
test set may be too easy or too difficult to classify and this can skew the results. On the other
hand, the instances in the test set may be valuable for training and when they are held out,
the prediction performance may suffer leading to skewed results.
To overcome this drawback in K-Fold Cross Validation, the available data is divided into k
equally sized folds. Subsequently, k iterations of training and validation are performed such
that, within each iteration a different fold of the data is held-out for validation while the
remaining (k-1) folds are used for training the classification model. Data is usually stratified
prior to being split into k folds i.e. data is rearranged to ensure that each fold contains
instances of all the classes in the problem at hand.
Leave-One-Out Cross Validation (LOOCV) is a special case of k-fold cross validation where k
equals the number of instances in the data. In other words, in each iteration, all the data
except for a single instance are used for training the model and the model is tested on that
single instance. An accuracy estimate obtained using LOOCV is known to be almost
unbiased but it has high variance.
To obtain more reliable performance estimates, multiple runs of k-fold cross validation can
be applied. The data is reshuffled and re-stratified before each round. This is referred to as
Repeated K-Fold Cross Validation.
2.6.2 Performance measures
In this section we review some of the most important measures to calculate classifier
performance. In particular we discuss the accuracy, sensitivity, specificity and precision
measures.
A classifier is tested by applying it to unseen test data with known classes and comparing
the predicted classes resulted from the classifier with the target classes.
The confusion matrix summarizes the correct and incorrect classifications resulted from a
given classifier. It displays both the actual target classes and the predicted classes. The
matrix dimension is M x M, where M is the number of classes of the problem at hand. The
entry m
ij
of such a matrix denotes the number of samples whose actual class is w
i
, and which
are assigned by the classifier to class w
j
.
Usually, in medical diagnosis, there are two classes, the positive class that indicates
infection/sickness and the negative class that indicates being healthy. For assessing the
performance of a given classifier, there are other important measures that need to be
considered in addition to accuracy. Among them are the sensitivity and the specificity.
Sensitivity is the proportion of correctly classified samples for being positive of all the
samples that are actually positive, while specificity is the proportion of correctly classified
samples for being negative of all the samples that are actually negative.
In the confusion matrix, in figure 1, A represents the number of samples that actually
belong to the positive class and are predicted to belong to the positive class. This is also

Confusion Matrix Predicted Classes
+ve -ve
Actual Target
Classes
+ve Class A B
-ve Class C D
Fig. 1. Confusion matrix.
quite often referred to as the true positive (TP). For medical application this would indicate
the number of patterns found to be sick; while they are really sick. B on the other hand
represents the false negatives (FN); i.e the number of sick people (positive samples) who have
been falsely classified to be healthy (or negative). Similarly C' is referred o as the false
positive (FP) while D indicates the true negative (TN).
Accuracy is the percentage of correctly classified samples. It can also be formulated as in
equation 4 using TP, TN, FP and FN.

TP TN
Accuracy 100%
TP FP TN FN
+
=
+ + +
(4)
Sensitivity measures the ability of a classifier to recognize the positive class (in our
application to detect sick people). It is also known as True Positive Rate (TPR) or recall.

TP
Sensitivity 100%
TP FN
=
+
(5)

On the other hand Specificity measures the ability of a classifier in detecting the negative
class (i.e healthy samples). This is also known as True Negative Rate (TNR).

TN
Specificity 100%
TN FP
=
+
(6)

The relationship between sensitivity and specificity, as well as the performance of the
classifier, can be visualized and studied using a receiver operating characteristic (ROC)
Curve. It is a graphical plot of the sensitivity (TPR) versus false positive rate (FPR) which is (1
specificity), for a binary classifier as its discrimination threshold is varied.
The ROC space is defined by two axes which are FPR and TPR representing the x-axis and
the y-axis, respectively. This depicts relative trade-offs between true positive representing
benefits and false positive representing costs. A point in the ROC space represents a
prediction of the classifier.
A prefect classification would yield a point in the upper left corner or coordinate (0, 1) of the
ROC space where there is 100% sensitivity (no false negatives) and 100% specificity (no false
positives). The point (0, 0) represents a classifier that predicts all cases to be negative, while
the point (1, 1) corresponds to a classifier that predicts every case to be positive. Point (1, 0)
is the classifier that is incorrect for all classifications as it means 100% false negatives and
100% false positives. The diagonal divides the ROC space. Generally, the points above the
diagonal represent good classification results while the points below the line poor results.

Precision is the proportion of the true positives against all the positive results.

TP
Precision 100%
TP FP
=
+
(7)

F
1
score (also F-score or F-measure) is a measure that considers both the precision and the
recall of the test to compute the score.

Precision Recall
F 2.
Precision Recall
=
+
(8)
This is also known as the F
1
measure, because recall and precision are evenly weighted. It is
a special case of the general F
measure (for non-negative real values of ).

( )
Precision Recall
2
F 1 .
2
.(Precision Recall)
= +
+
(9)
Two other commonly used F measures are the F
2
measure, which weights recall higher than
precision, and the F
0.5
measure, which puts more emphasis on precision than recall. Usually
the measure chosen for performance evaluation is application dependent. In medical
applications usually precision and recall in addition to accuracy- are very important. In
our case study presented in section 4 we use accuracy, sensitivity, specificity and precision
to evaluate the proposed model for DNA microarray data classification.
3. Machine learning techniques in bioinformatics
Due to the availability of huge amounts of data delivered by high-throughput
biotechnologies, data management procedures are required to provide the ability to store
and retrieve biological information efficiently (Valentini, 2008)( Goble & Stevens,2008); this
is in addition to the need of methods to extract and model biological knowledge from the
data (Baldi & Brunak, 2001).
Machine learning techniques deal with a wide range of bioinformatics problems in
genomics, proteomics, gene expression analysis, biological evolution, systems biology, and
other relevant bioinformatics domains (Valentini, 2008) (Larranaga et.al., 2005). As follows
we briefly review the use of machine learning in each of the previously mentioned fields of
bioinformatics. However, the rest of the chapter will focus on micro-array data classification.
The state of a cell consists of all those variables-both internal and external-which determine
its behaviour. According to the Central Dogma of molecular biology, the activity of a cell is
determined by which of its genes are expressed i.e., which genes are ``turned on", resulting
in the active production of the respective proteins. When a particular gene is expressed, its
DNA is first transcribed into the complementary messenger RNA (mRNA), which is then
translated into the specific protein this gene codes for. We can measure the level of
expression of each gene (i.e. how much each gene is ``turned on") by measuring how many
mRNA copies are present in the cell (Lander, 1996).
Genomics is one of the most important domains in bioinformatics. It studies biological
sequences at genome level such as DNA and RNA. (Mathe et al., 2002) provide a review on
some important applications which are locating the genes in a genome and identifying its
function. Ensemble methods have been applied to predict gene function in comparison with

single classifier as in (Re & Valentini, 2010), where several data sources are integrated then
input to SVM base classifiers and combined using weighted average and decision templates.
The ensembles outperform the single SVM classifier. Sequence information is also used for
gene function and RNA structure prediction (Freyhult, 2007) as well as many other relevant
genomics problems.
Gene expression data analysis is a well-established bioinformatics domain where Machine
Learning methods for classification and clustering have been widely applied. DNA gene
expression microarrays allow biologists to study genome-wide patterns of gene expression in
any given cell type, at any given time, and under any given set of conditions (Baldi &
Brunak, 2001). Gene expression data is arranged into a matrix where, columns represent
genes and rows represent the samples. Each element in the matrix represents the expression
level of a gene under a specific condition and it is represented by a real number.
The use of these arrays produces large amounts of data, potentially capable of providing
fundamental insights into biological processes ranging from gene function to development,
cancer, aging and pharmacology (Baldi & Brunak, 2001). However the data needs to be pre-
processed first, i.e. modified to be suitably used by machine learning algorithms. Then the
data is analyzed to look for useful information.
Clustering techniques such as k-means, hierarchical clustering (Eisen et al., 1998) and self-
organizing maps (SOMs) (Tamayo et al., 1999) have been applied to identify genes
according to their function similarities. These methods assume that related genes have
similar expression patterns across all samples and hence divide the set of genes into disjoint
groups. Accordingly, identifying local patterns with subset of genes that are similarly
expressed over a subset of samples is difficult using traditional clustering techniques.
(AboHamad et al., 2010) propose a bi-clustering technique which is based on clustering
similarly expressed genes set over a subset of samples simultaneously. On the other hand,
many classification techniques are used. The majority of papers published in the area of
machine learning for genomic medicine deal with analyzing gene expression data coming
from DNA microarrays, consisting of thousands of genes for each patient, with the aim to
diagnose (sub) types of diseases and to obtain a prognosis which may lead to individualized
therapeutic decisions (Bellazi & Zupan, 2008). The published papers are mainly related to
oncology, where there is a strong need for defining individualized therapeutic strategies
(Mischel & Cloughesy, 2006). A seminal paper from this area is that of (Golub et al., 1999)
and focuses on the problem of the early differential diagnosis of acute myeloid leukemia
(AML) and acute lymphoblastic leukemia (ALL). Several classification techniques have been
applied on different benchmark datasets, among these are decision trees, nave bayes
classifier, multilayer perceptron and SVMs which have proved to be very effective in such
applications. The mentioned classification approaches are usually coupled with feature
(gene) selection methods to improve the performance. To avoid removing some features, the
use of ensembles has emerged with different ways of distributing features among subsets as
explained in section 2.
Proteomics is the field that studies proteins. Proteins transform the genetic information into
actions performed in life. The prediction of the secondary and tertiary structure of proteins
represents one of the main challenges for Machine Learning methods in bioinformatics.
Neural networks have been applied to predict protein secondary structure (Baldi & Brunak,
2001). This is due to the fact that proteins are very complex macromolecules with thousands
of atoms and bounds so there are huge number of possible structures. This makes protein
structure prediction a very complicated combinatorial problem where optimization

techniques are required. Machine learning is also applied for protein function prediction,
fold recognition as well as other relevant proteomics problems (Valentini, 2008).
Systems biology is an emerging bioinformatics area where Machine Learning techniques play
a central role (Kitano, 2002). It is concerned with modelling biological processes inside the
cell. Mathematical models and learning methods are required to model the biological
networks ranging from genetic networks to signal transduction networks to metabolic
pathways (Bower et al., 2004).
Phylogenetic trees are schematic representations of organisms' evolution. Machine Learning is
applied for phylogenetic tree construction by comparisons made by multiple sequence
alignment where many optimization techniques are used (Larranaga et al., 2005).
As follows we present a novel machine learning model for micro-array data classification.
Experiments and comparative results demonstrate the efficiency of these models to deal
with high-dimensional DNA data.
4. A case study of an SVM ensemble using feature subset selection for DNA
classification
In this section, we present a case study of a SVM ensemble that uses SVM base classifiers
and another SVM classifier for combining the results of the base classifiers to get the final
classification. The proposed ensemble uses k-means clustering for grouping the features into
subsets. The ensemble is referred to as k-means-SVM fusion throughout the rest of this chapter.
The flow charts in figures 2, 3 and 4 illustrate the main phases used for building the
ensemble. A dataset consisting of a set of labelled examples is initially given. Each example
is characterized by a set of features and a label indicating its class. The dataset is divided
into a training set, a validation set and a test set. For clustering, the features are grouped into
k feature subsets and k-means clustering is applied to the training set. Then, each of the SVM
base classifiers is trained using the training set characterized by features of a single feature
subset. The SVM classifier responsible for fusion is trained using the validation set and then
the ensemble is ready to be tested on the test set.
Figure 5 presents the steps of the algorithm for building the ensembles that use an SVM
classifier for fusion in more details. Initially, the available data set Z containing all features is
divided into training Z
Train
, validation Z
Valid
and testing set Z
Test
. The training set Z
Train
is used
for building the base SVM classifiers and determining their parameters through cross
validation by further splitting the training set into training part and validation part, applying
grid search using range of values for the parameters and selecting the parameter values that
resulted in the best accuracy among the validation set. The validation set Z
Valid
is used to train
the combiner SVM, while the test set Z
Test
is used to evaluate the overall ensemble.
Before any of the data parts are applied, we first perform a feature subset selection
procedure to choose k subsets to be used as an input for each base classifier. The input to
any base SVM classifier i is hence the training samples with features in the cluster i. After
the base classifiers are trained using the training portion Z
Train
, the combiner is trained using
the validation set Z
Valid
as follows:
The outputs of the k SVMs are collected to form a new feature-sample training matrix for the
SVM combiner where each sample is characterized by the base SVM outputs as features and
its label is the same as its original labels.
In the test phase, the overall accuracy of the ensemble is tested using the remaining samples
of Z
Test
reserved.


Fig. 2. Ensembles using SVM fusion.
Cross validation is used to evaluate the proposed model using different
partitions of Z for training and validation.
Train SVM
1

using the feature
subset FS
1
of the
training set
Train SVM
2
using
the feature subset
FS
2
of the training
set

Train SVM
k
using
the feature subset
FS
k
of the training
set

Partition the features into k disjoint feature subsets chosen using
k-means clustering applied to the training set
FS
1
, FS
2
FS
k
Train the SVM Combiner
1
Test the whole ensemble on
the test dataset
2

Dataset
End
Start


Fig. 3. Training the SVM Combiner in the Ensemble.
Train the SVM Combiner using the outputs of the
base classifiers on the validation dataset
Get the k feature subsets (FS
1
, FS
2
FS
k
)
Test SVM
1

using the
feature subset
FS
1
of the
validation set
Test SVM
2

using the
feature subset
FS
2
of the
validation set
Test SVM
k

using the
feature subset
FS
k
of the
validation set

Dataset (Validation set)
1
End


Fig. 4. Testing the ensemble with SVM fusion.
5. Data and experimental setup
In this section, we describe the experiments conducted to test and evaluate the proposed the
k-means-SVM fusion ensemble on the Leukaemia data set. Refer to (Ahmed et al., 2010) for
more experiments on other data sets.
The Leukemia dataset is a benchmark micro-array dataset which consists of 72 samples,
7129 features and 2 classes (AML and ALL). The 72 samples consist of 47 samples of Acute
Lymphoblastic Leukemia (ALL) and 25 samples of Acute Myeloblastic Leukemia (AML).
The training and test samples in (Chang & Lin, 2001), (Golub et al., 1999) are merged then
normalized as indicated in (Shevade & Keerthi, 2003).
The proposed k-means-SVM fusion ensemble is compared to a single SVM classifier as well
as three different SVM ensembles: the random-majority vote ensemble, the k-means-majority
vote ensemble and the random-SVM fusion ensemble. The random-majority vote ensemble uses
the random subspace method to select feature subsets and distribute them among base
classifier in the ensembles. A fixed fusing rule majority vote- is used to combine the output
of the base classifiers. In contrast, the k-means-majority vote ensemble uses feature subsets
resulting from k-means clustering as described in section 4 but still uses majority voting for
Test SVM
1

using the
feature
subset FS
1
of
the test set
Test SVM
2

using the
feature subset
FS
2
of the test
set
Test SVM
k

using the
feature subset
FS
k
of the test
set
Test the SVM Combiner using the outputs of the
base classifiers on the test data.
Get the k feature subsets (FS
1
, FS
2
FS
k
)
Dataset (Test set)
2
End

classifier fusion. Alternatively, the random-SVM fusion ensemble uses Random subspace
method for feature subset selection and a trainable SVM classifier as a combination rule.

Given:
Z, a set of N crisp labelled samples x.
FS: (1 n), a set containing all n features.
Step 1: Choose feature subsets using k-means clustering
k-means on dataset Z
Train

Initialize k, the number of clusters and the number of the base classifiers.
Cluster analysis on features using Z
Train
using k-means algorithm.
Get $k$ disjoint feature subsets FS
1
, FS
2
, , FS
K
.
Step 2: Train the Base Classifiers
Train the base classifiers using Z
Train
such that each base classifier SVM
i
is trained
with feature subset FS
i
.
Step 3: Train the Combiner
Test every SVM (1 k) base classifiers using Z
Valid
with the corresponding feature
subset FS(1 k).
Train the SVM(combiner) using the outputs of the SVM(1 k) on the validation data
set Z
Valid
.
Step 4: Test the ensemble
Test the ensemble using the Test data set Z
Test
as follows:
Z
Test
is passed through the SVM (1 k) base classifiers.
Z
Test
with the outputs of the SVM base classifiers as features is given to the
SVM(combiner).
SVM(combiner) classifies the samples of Z
Test
.
Fig. 5. Ensembles using SVM fusion.
The suggested ensembles are tested for different number of feature subsets, i.e different
number of base classifiers. Experiments are repeated for number of feature subsets k = 2
n
-1
where n = 2, 3, 4, 5, 6. Results are compared using different measures of performance
including accuracy, sensitivity, specificity and precision. For the sake of brevity we only
present results based on accuracy and sensitivity.
Cross validation is used to obtain different training, test and validation sets. Since DNA
microarrays are characterized by having a very small number of samples, the training and
validation sets are overlapped with 1/3 of the samples.
The usage of cross validation differs according to the ensemble model. For the ensembles
that use majority vote combiner, the dataset is divided into a training set and a test set. The
training set is used to train the base classifiers while the test set is used to test the base
classifiers then the majority vote is applied to their outputs. For tuning the parameters of the
base classifiers, the training set is further split into a training set and a validation set on
which the classifier is validated. Grid search using range of values for the parameters is
applied and the parameter values that get the best performance on the validation set are
chosen for the base classifiers.
For the ensembles that use a SVM for fusion, the dataset is divided into a training set,
validation set and a test set using k-fold cross validation. The training set is used to train the
base classifiers then the validation set is used to test the base classifiers. The outputs of the

base classifiers are then used in the training of the SVM combiner. The test set is then used
to test the whole ensemble.
All experiments are performed using LibSVM (Chang & Lin, 2001) using 5 fold cross
validation for training the base classifiers and the combiner. K-means is applied with values
for k ranging from 3 to 63 with increment of 2. Linear kernels are chosen for the SVM base
classifiers as well as for the SVM combiner as it was found in literature that they are suitable
for the high dimensional microarray datasets (Chang & Lin, 2001), (Bertoni et al., 2005). For
each SVM with linear kernel, parameter C requires to be optimized. This is done using grid
search by 5 fold cross validation.
We experimented with exponentially growing sequences of C in the rage of -15 to 15 to
identify a good value for the parameter.
6. Results and discussions
Figure 6 compares the accuracy of the k-means-SVM fusion ensemble to the random-majority
vote ensemble, the k-means-majority vote ensemble and the random-SVM fusion ensemble for
different number of feature subsets (i.e. different number of base classifier or ensemble
sizes).

Fig. 6. Test accuracies of the four ensembles with respect to the number of feature subsets.
The accuracy of the K-Means-SVM fusion ensemble seems to outperform the other models
with growing number of feature subsets (i.e. with increased number of base classifiers).
Also, the accuracy of random-SVM fusion increases with higher number of feature subsets.
However, it still has the lowest accuracy among the four ensembles.

On the other hand, for the k-Means-majority vote ensemble, increasing the number of feature
subsets results in a drop of the accuracy; while for random-majority vote ensemble results are
not affected by the change of the number of feature subsets.
Table 1 summarizes the best results obtained for each ensemble across the different number
of feature subsets in addition to those obtained using a single SVM classifier. The number of
feature subsets at which the best results are obtained is mentioned for each ensemble. It can
be noticed that the ensembles that use majority vote combiner work well only with small
number of feature subsets while those that use an SVM classifier for fusion need a large
number of feature subsets.
Results reveal that the k-means-SVM fusion ensemble outperforms k-means-majority vote
ensemble as well as random-SVM fusion and random-majority vote ensembles. K-means-SVM
fusion ensemble also shows to have a high sensitivity with respect to the other ensembles in
the comparison.
Since for the leukemia dataset, both classes are patients, there is no positive or negative
class. Accordingly, the sensitivity is calculated twice; at first, considering AML as the
positive class and then considering ALL as the positive class. The average of both is then
calculated.

Classifier/Ensemble Accuracy Sensitivity (Average)
Single SVM classifier 92.86 15.97 81.68
Random-Majority Vote (3) 92.86 15.97 90.00
Random-SVM Fusion (41) 87.14 19.17 84.67
K-Means-Majority Vote (3) 92.86 15.97 90.00
K-Means-SVM Fusion (63) 97.14 3.92 96.89
Table 1. Best classification accuracy and sensitivity measures obtained by applying the
ensembles and the single SVM classifier to the leukemia dataset. The number of feature
subsets at which the best results are obtained are mentioned between brackets.
Figures 7-10 illustrate for each ensemble the improvement of the combined model over the
average performance of the base classifiers. The figures show the average accuracies of the
base classifiers of each ensemble compared to the ensemble accuracies. In addition the ratio
of the ensemble accuracies to those of the base classifiers are depicted. It is obvious that the
k-means-SVM fusion has the best ratio among the four ensembles.
Figure 7 shows the results for the k-means-majority vote ensemble. It can be noticed that the
ensemble improves the performance of the base classifiers but its accuracy drops with
higher number of the feature subsets. So, it works better with small number of feature
subsets. Figure 8 demonstrates the performance of the k-means-SVM fusion ensemble. Clearly
the ensemble enhances the performance of the base classifiers except when using 3 feature
subsets. Unlike the k-means-majority vote ensemble, its performance does not drop with
increased number of feature subsets. K-means-SVM fusion ensemble achieves the best
accuracy among the four ensembles when using 63 feature subsets. Figure 9 summarizes the
performance of the random-majority vote ensemble. It is noticed that it has a slight
improvement over the average performance of the base classifiers resulting in a nearly
constant behaviour. Figure 10 demonstrates that random-SVM fusion ensemble does not

work well on using a small number of feature subsets but as the number of feature subsets
increase, it improves the performance and become better than the average performance of
the base classifiers.

Fig. 7. Results of the k-means-majority vote ensemble.

Fig. 8. Results of the k-means-SVM fusion ensemble.


Fig. 9. Results of the random-majority vote ensemble.

Fig. 10. Results of random-SVM fusion ensemble.
As a general conclusion of the previous experiments we can state that the ensembles with
SVM classifier as base classifiers generally improve the classification accuracy over single
classifiers. Ensembles that use an SVM classifier for fusion outperform those that use
majority vote as a combiner when using a reasonably large number of feature subsets and
base classifiers.

According to the study on the leukemia dataset, k-means-SVM fusion ensemble performs
the best among the four ensembles with regards to both accuracy and sensitivity. More
results to confirm this conclusion are reported in (Ahmed et al., 2010).
7. Conclusions and future directions
This chapter presents a broad introduction to machine learning and focuses on the
classification problem in bioinformatics. In particular we cover main terminologies from the
pattern recognition, machine learning and data mining fields. We try to review main models
used for classification and to elaborate on classifier testing and evaluation techniques. We
devote a special attention to SVM, ensemble techniques and feature subset ensembles as
they are the base of our proposed DNA micro-array data classification model. The proposed
classification model exploits the use of powerful machine learning models such as SVMs
and ensemble methods coupled with feature subset selection. The proposed approach
proves to be able to deal with data challenges that are imposed by this application which is
mainly the huge number of features and the small samples size.
Results are shown on the leukemia dataset and compared to four different models. The
study concludes that the use of ensembles is very fruitful in such applications. The way of
distributing the features among subsets affects the performance of the ensemble. K-means is
a systematic way that proved to be suitable for clustering the features into subsets especially
when used with a SVM classifier for combination. For the leukemia dataset, k-means-SVM
fusion ensemble performed the best with respect to accuracy and sensitivity. The study
confirms the importance of ensembles in bioinformatics applications and highlights that the
coupling between the method of distributing the features among subsets and the
combination method is crucial for obtaining good results.
Different method can be investigated for distributing the features among subsets. Higher
numbers of base classifiers / numbers of feature subsets can be experimented with. Time
complexity of the proposed models need to be calculated and accessed. The use of other
combiners especially classifiers are worth investigating. In addition, the use of the proposed
models can be extended to other data sets and other domains in the bioinformatics field.
8. Acknowledgment
This work was supported by DFG (German Research Society) grants SCHW 623/3-2 and
SCHW 623/4-2.
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Part 9
Next Generation Sequencing

29
Deep Sequencing Data Analysis:
Challenges and Solutions
Ofer Isakov and Noam Shomron
Sackler Faculty of Medicine,
Tel Aviv University,
Israel
1. Introduction
Ultra high throughput sequencing, also known as deep sequencing or Next Generation
Sequencing (NGS), is revolutionizing the study of human genetics and has immense clinical
implications. It has reduced the cost and increased the throughput of genomic sequencing
by more than three orders of magnitude in just a few years, a trend which is guaranteed to
rapidly accelerate in the near future (Metzker, 2010). Using deep sequencing, for example, it
is now possible to discover novel disease causing mutations (Ley et al., 2008) and detect
traces of pathogenic microorganisms (Isakov et al., 2011). For the first time, research fields
such as personalized medicine for patient treatment are becoming tangible at genomic levels
given advances in deep sequencing data integration.
The amount of data produced by a single ultra high throughput sequencing run is often
tremendous and can reach hundreds of millions of reads in various lengths per experiment
(Mardis, 2008). The storage, processing, querying, parsing, analyzing and interpreting of
such an incredible amount of data is a significant task that holds many obstacles and
challenges (Koboldt et al., 2010). In this chapter we will address some of the possibilities,
potentials and questions raised during ultra high throughput sequencing data analysis. We
will mainly focus on common pre-analysis concepts and crucial advanced considerations for
alignment, assembly and variation detection. Currently, the deep sequencing user is faced
with an abundance of deep sequencing data analysis tools, both publicly and commercially
available. For each of the aforementioned analysis types, we will point out the various
aspects to be considered when choosing a tool, and emphasize the relevant challenges and
possible limitations in order to assist the user in picking the most suitable one. Since deep
sequencing data analysis is a rapidly evolving field, our focus will be on fundamental
concepts of the analysis process and the its challenges, allowing this read to be relevant
amid additional published software.
Our first part will encompass a brief overview of current leading deep sequencing
technologies with special attention to their features, strengths and possible drawbacks in
regards to the different preliminary questions that one might ask when using ultra high
throughput sequencing. The second part of the chapter introduces pre-analysis processes.
These are common quality control and assurance methods that alleviate deep sequencing
derived biases and improve the overall results of any down-stream analysis. In the third
part of the chapter, we will go over the different aspects of the post-sequencing analysis,


656
specifically deep sequencing data alignment, assembly and variant detection. For each
section we will cover leading methods and tools, quality evaluation and filtration and
address the requirements, capabilities and limitations of these tools. The section on variation
detection will cover both common variant detection considerations, variant specific
challenges and currently available solutions.
2. Sequencing technologies
Sequencing technologies are evolving rapidly, with an overwhelming increase in efficiency
and throughput (Mardis, 2008). This expeditious rate of change and improvement is
accompanied by a variety of different sequencing platforms, with both great similarities and
differences alike. Without going into the technology underlying each sequencing platform in
detail, we will specify advantages and limitations both general and specific, that are relevant
for deep sequencing experiment design. For this purpose, we will refer to the leading
commercially available platforms produced by Roche/454 (Margulies et al., 2005a),
Illumina/Solexa (Bentley et al., 2008), Life/APG (SOLiD) (McKernan et al., 2009) and Pacific
Biosciences (Eid et al., 2009).
The initial step in the sequencing process is random fragmentation of the nucleotide sequence
of interest, in order to increase throughput by simultaneously sequencing millions of
fragments. These template fragments can then either undergo clonal amplification, in which
they are ligated with adapters and amplified using common PCR (Polymerase Chain Reaction)
primers (Roche; Illumina; Life), or they can be used as the sequencing templates themselves
(single molecule templates; Pacific Biosciences). Clonal amplified template preparation
requires a higher amount of initial DNA material. Since this technique relies on PCR
amplification, errors might be introduced to the target before the sequencing process begins.
The amount of introduced errors is related to the fidelity of the polymerase utilized in the
reaction (Chan, 2009). These potential background errors could be considered actual sequence
variants in the down stream analysis. PCR utilization might also result in amplification bias,
misrepresenting high GC content areas. Such is the case in a recent study in which PCR
introduced expression biases for GC rich chromosomes required additional assessment,
hampering the uniformity of the results (Chiu et al., 2010). Simultaneously sequencing clonal
amplified templates is further complicated by potential different extension rates that cause
asynchronous sequencing (phasing), resulting in a higher background noise. Single molecule
template sequencing (Schadt, Turner, & Kasarskis, 2010) does not require PCR amplification
thus circumventing its derived amplification and clonal sequencing biases making it an
appropriate tool to be used in quantification experiments (e.g RNA-seq, Chip-seq etc.) and in
cases where the initial sample DNA content is scarce. Because sequencing is performed on a
single molecule and sequences are inferred from extremely weak signals, the correcting effect
of simultaneous same-sequence template sequencing is lost resulting in a higher error rate
(Schadt et al., 2010). Therefore a higher sequencing fidelity is required (Metzker, 2010).
In addition to the aforementioned general template preparation and sequencing method
associated biases, one needs to consider the inherent benefits and shortcomings of each
sequencing technology. Pyrosequencing, for example, employed by the Roch 454's GS FLX
platform, generates long reads (~400nts) and presents relatively unbiased coverage,
enhancing de-novo genome assembly and improving alignment capabilities, thus making it
an appropriate tool for SNP and structural variations discovery, demonstrating low false
positive rates (Margulies et al., 2005a; Nothnagel et al., 2011). However, the technology's

Deep Sequencing Data Analysis: Challenges and Solutions

657
susceptibility to insertion and deletion errors and higher rate of homo-polymer (e.g
contiguous run of the same base pair) sequencing errors should be considered when
performing a variation oriented research (Chan, 2009). Current reverse termination
(Illumina's Genome Analyzer or HiSeq 2000) and sequencing by ligation (Life's SOLiD)
technologies produce shorter reads (<200nts) but at a much higher throughput and are
considered optimal for small scale variants detection (e.g SNPs and indels) due to very high
detection resolution owed to massive read overlap and high coverage. However, short
reads' inherent problems of ambiguous mapping and complicated assembly can result in
higher false positive rates in variant discovery (Nothnagel et al., 2011), that could be
alleviated by higher throughput and employment of paired-end sequencing (e.g sequencing
both ends of a fragment template) (Medvedev et al., 2009; Metzker, 2010). Sequencing by
ligation technology, employed by Life's SOLiD, reads the colors of fluorescently marked
ligated primers and converts them into the template sequence. SOLiD is less susceptible to
phasing errors and the unique conversion of color to sequence results in an inherent error
correction and thus a more accurate SNP detection process. However, this reduced error
rate requires utilization of a reference genome in the color conversion process (Kircher &
Kelso, 2010). We also note that error rate increases across all platforms towards the end of a
sequenced read (Dohm et al., 2008), due to reduced enzyme efficiency, loss of enzymes,
increased phasing effect or incomplete dye removal. The different attributes of the variety of
platforms can result in significantly different output data and performance, and it was
demonstrated that the combination of more than one platform is potentially more cost
effective and could yield higher fidelity and accuracy (Dalloul et al., 2010; Nothnagel et al.,
2011). We will now discuss how to alleviate some of these inherent and general difficulties
using pre-analysis processing.
3. Pre-analysis processing
In this section we will discuss the processing performed on deep sequencing output data
prior to the specific experimental analysis. Mentioned above are examples of the vast cross
platform differences that could affect the downstream analysis and thus the biological
conclusions derived. These differences accompany the inherent bias in deep sequencing
experiments (Dohm et al., 2008; Schwartz et al., 2011). In order to reduce these possibly
confounding effects, platform manufacturers and developers provide the end-user with a
sequencing quality scale for both automated and recommended manual quality based data
filtration and refinement (Bentley et al., 2008; Harris et al., 2008; Margulies et al., 2005a; K. J.
McKernan et al., 2009). We will suggest quality confirmation methods for the text based
output end-users face after a sequencing run, and discuss common necessary pre-analysis
processing steps that ensure data validity and proper utilization. For each platform's
inherent quality assurance and control measures, one should address the specific platform's
technical support and annotation.
The most common initial form of output format is either a sequence FASTA file
accompanied by a numerical quality QUAL file, describing the per-base probability of
incorrect sequencing based on the PHRED quality score (Ewing and Green, 1998; Ewing et
al., 1998), or the FASTQ format (Cock et al., 2010), containing sequences coupled with their
quality stored as ASCII characters. Currently, Sanger FASTQ files use ASCII 33126 to
encode PHRED qualities from 0 to 93 (i.e. PHRED scores with an ASCII oset of 33),
marking an error probability between 10
0
and 10
-93
. Up until the Genome Analyzer v1.3,


658
Illumina utilized a different scoring scale in their sequencing output, described in (Cock et
al., 2010). Currently Illumina encodes PHRED scores with an ASCII oset of 64, and so can
hold PHRED scores from 0 to 62 (ASCII 64126). Life's SOLiD produce a color based FASTQ
file (CSFASTQ) that utilizes the digits 0-3 to mark the sequenced color, the processing of
which we will not cover in this section. Though these different scoring methods potentially
contribute to misinterpretation and confusion, they can be easily converted and conformed
(Cock et al., 2009; Goto et al., 2010; Holland et al., 2008; Stajich et al., 2002). Most current
analysis tools are able to handle both scoring methods, though some require specific
parameters to be set for dealing with each. When employing these analysis tools, one should
mind the appropriate quality score is used.
Quality control of deep sequencing data refers to an overview on the base and quality
distribution between lanes, tiles and cycles, and correlating the initial sequence data with
expected length, GC content, ambiguous bases, sequence complexity and alignment ensuing
location distributions which can hold information regarding possible sequencing bias,
contamination or artifacts. Platform specific quality control tools.
(Cox et al., 2010; Dolan and Denver, 2008; Martinez-Alcantara et al., 2009) and more general
quality assessment software (Dai et al., 2010; Schmieder and Edwards, 2011) can help
circumvent such biases, by both raising awareness to implicating irregularities with textual
and graphical data representation and by removing such low quality or aberrant sequences
prior to the downstream analysis. The need for careful quality control is exemplified by
deep sequencing data with a tile specific A base bias, leading to over-expression of the base
in the sequences derived from that tile. When searching for rare sequence variants, such
base over-expression should be considered when sequences supporting an A variant are
derived from the aforementioned tile. A more common example is sequence duplication
(Gomez-Alvarez et al., 2009),usually an artifact of PCR amplification and other library
preparation processes, that cause over-representation of certain sequences. This creates a
skewed coverage distribution that may subsequently bias the error model and thus
substantially increase the number of false-positive SNP discoveries and tilt expression and
metagenomic analysis results. Available quality control software allow the user to
completely remove these duplicates (FASTX - toolkit; Li et al., 2009) or mark them for
downstream analysis consideration (PICARD). Recently various algorithms utilizing suffix
tree data structures were developed for sequencing error correction (Kelley et al., 2010; Zhao
et al., 2010).
A common procedure in the pre-analysis process, following initial quality control, and prior
to sequence duplication removal, is the compulsory tag / adapter removal (Lassmann et al.,
2009; Schmieder et al., 2010) and optional quality trimming. Tags are used during the library
preparation phase for amplification or differentiation processes (e.g multiplexing; Galan et
al., 2010). If they are sequenced, they can profoundly affect the downstream analysis unless
removed (e.g clipping). The clipping process, removes any tag remnants from the sequence
reads, ridding the data from reads composed mainly or even solely of the tags. The user
must set the minimal read length to be retained (according to the sequenced sample and
experimental question) and consider possible sequence similarities between the sample and
the adapters. Trimming, refers to the sequence removal from either the 5' or the 3' ends of a
read where either the sequence complexity or quality does not pass user settings. It is often
used for poly-A or poly-T removal, or removal of bases with significantly lower, bias
introducing quality scores. Unlike clipping, which is mandatory for valid downstream
analysis, trimming is only recommended to improve accuracy and performance in


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subsequent analysis steps such as alignment and assembly. Following both clipping and
trimming, the researcher may review the sequence data for size distribution, and verify
concordance with the experimental context. For example, when performing microRNA
sequencing experiments, one would expect the sequence size composition to be
approximately 20-24 nts in length. If the majority of the data deviates from this range, a
more careful examination of the information is in order and library preparation bias should
be considered.
We urge the user to consider the sequencing data in the appropriate experimental context
and utilize the aforementioned quality control and assurance methods prior to the
downstream analysis to increase the experimental validity and accuracy and to ensure
better, more reliable results.
4. Data analysis pathways
In the previous sections, we covered common deep sequencing data considerations and
refinement, crucial and beneficial for all types of down stream analysis. In this section, we
will go over the common data analysis pathways and possibilities, covering their
appropriate utilization, the benefits and limitations of each pathway, and familiarizing the
user with some of the common available analysis tools.
4.1 Alignment
Most of the analysis pathways specified below involve an initial step of mapping the deep
sequencing reads against a reference genome of either the sequenced species, or a related
organism with sufficient genetic resemblance. This step presents a computational challenge
due to the sheer amount of short reads produced in deep sequencing experiments. It is
further complicated by nucleotide and structural variance, sequencing errors, RNA editing
and epigenetic modifications. When deep sequencing was initially introduced, established
early-generation sequence alignment tools (Altschul et al., 1990; Kent, 2002) more suited for
the query of a limited number of sequences were less appropriate for high throughput
sequencing's millions of short sequence fragments mapping (Trapnell and Salzberg, 2009),
requiring novel alignment algorithms and tools to be specifically designed. Current short
read alignment tools.
(Langmead et al., 2009; Li and Durbin, 2009; Li et al., 2008; Li et al.,2009; Lin et al., 2008;
Novoalign), utilize various heuristic techniques for alignment of millions of short sequences
within an acceptable time requirement (Flicek and Birney, 2009). This section will not cover
the underlying algorithms for each tool (Li and Homer, 2010). Instead, we will address a few
imperative features to be considered when initiating data analysis and alignment.
When choosing an alignment tool, one needs to consider the memory and time requirements
and limitations and the appropriateness of the tool to the exploratory question at hand.
Some important features to be considered include:
Quality utilization and control - As we mentioned before, sequencing quality provides the
user with initial assessment of the data. Some alignment tools, utilize these quality scores
(Langmead et al., 2009; Li et al., 2008; Novoalign) and it was shown that such employment
greatly improves the mapping performance (Frith et al., 2010; Li and Homer, 2010). Most
common alignment software generate the alignment output in the Sequence Alignment Map
(SAM) format (Li et al., 2009), with a multitude of supporting downstream analysis tools.
This common format provides users with a simple and flexible common ground to evaluate


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alignment results and easily extract and utilize data for further analysis. As for the
sequencing output, so does the alignment output contain a PHRED based quality score for
each of the aligned reads, describing the probability of per-base false alignment.
Combination of this quality score together with other alignment parameters such as
mismatches could and should be further assessed using specialized tools (Lassmann et al.,
2011) in order to characterize mapped and unmapped reads for potential alignment
improvement. These alignment quality scores can be re-assessed using currently available
tools (McKenna et al., 2010; Novoalign), so that they better denote the probability of a
mismatch between the aligned base and the reference sequence. This quality recalibration
takes into account the given base and its quality score, the position within the read and the
adjacent nucleotides to account for sequencing chemistry biases (Li, Li et al. 2009), and was
shown to reduce the effect of sequencing technology derived biases and improve overall
variant detection fidelity (DePristo et al., 2011).
Gapped alignment An important feature one should be mindful of when choosing an
alignment tool is whether the tool utilizes the gapped alignment algorithm. Since gapped
alignment only mildly increases alignment sensitivity, it is not crucial to pick a supporting
tool for many general purposes. However it is especially crucial for variant calling,
specifically insertions and deletions (indels) detection (Krawitz et al., 2010) and it is highly
recommended to choose a tool that implements gapped alignment (Li and Durbin, 2009; Li
et al., 2008; Novoalign), when venturing on variant detection experiments, or when targeting
known indel abundant areas.
Mismatches and Gap penalties Most alignment tools allow the user to set the number of
allowed mismatches between the read and a reference location and the scoring scale for gap
opening and extension. Allowing more mismatches results in a higher portion of mapped
reads but at the cost of increased ambiguity and reduced confidence of these alignments.
Mismatch allowance should be set while considering the specific experiment at hand. For
example, when undergoing microRNA expression profiling, one will want an accurate
estimate of the abundance of each microRNA , and should not allow a high mismatch rate if
any. On variant calling experiments however, the user should consider the possible
expected size range of the variants before setting the allowed mismatch and gap penalty
parameters (e.g, if one aims to find a >5nt long deletion, the mismatch limitation should
allow it).
Multiple mapping - In theory, unique alignment, mapping a read to a single unique loci on
the reference genome is expected by most reads longer than 30 nts when aligning against a
large human scale reference. Usually, a portion of the reads will remain unmapped due to
contaminant origin or sequencing errors, or more commonly, they will ambiguously map to
several different locations (multiple mapping) due to sequence homology and
repetitiveness. Different alignment tools flag these multiply mapped reads, and provide the
user with the option to either randomly assign them to one loci (Li and Durbin, 2009) or just
output all of them (Novoalign). Researchers may choose to incorporate only uniquely
mapped reads into their downstream analysis, or set a maximal number of different
mapping locations for incorporated reads. Discarding multiply mapped reads results in loss
of a substantial portion of the data, with potential crucial effects on the following analysis.
Currently, there are several approaches for allocation of these multiply mapped reads. One
method is to count each read as if originating from each of the mapped loci, potentially
over-estimating the expression or coverage of some, since the same read could not have
originated from more than one loci. Another method is to divide each read count between


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all its mapped loci, adding a small equal portion to each. This could have the opposite effect
of under-estimating expression and coverage, especially for low complexity loci. Several
methods utilize heuristics for dividing these reads amongst their mapped loci according to
the uniquely mapped reads in those regions (Hashimoto et al., 2009; Mortazavi et al., 2008).
A fairly novel approach utilizes probabilistic models such as maximum likelihood to
compute the most likely origin of each read greatly improving the results of quantitative
deep sequencing experiments and differential expression (Paaniuc et al., 2011).
Since each parameter can greatly affect various performance attributes, considering the
aforementioned features is crucial when initiating deep sequencing data alignment. The user
should always mind the alignment tool's inherent limitations and implement parameters
settings according to the experiment at hand and the expected possible downstream
analysis, picking an appropriate tool and tuning necessary features for optimal alignment
results.
4.2 Assembly
Assembly refers to the process of piecing together short DNA/RNA sequences into longer
ones (e.g contigs) which are then grouped to form scaffolds for computationally
reconstructing a sample's genetic component. When the assembly process is performed with
the assistance of a reference genome, it is referred to as mapping assembly, if no reference is
available it is called de novo assembly. Original computational assembly tools were designed
to use capillary-based sequencing's 800 base pairs long sequences in order to deduce the
original full sequence through examination of overlapping segments. Deep sequencing data
presents a more compound assembly problem due to higher amounts of sequences that are
significantly shorter. Though it adds complexity to the process, this significant increase in
throughput enables the successful realization of whole mammalian genome de novo
assembly as shown in (Li, Fan, et al., 2010; Li, Zhu, et al., 2010). Sequencing errors, uneven
genome coverage and reads too short to be informative in repeated regions required a new
breed of assembly tools designed specifically for short reads (Butler et al., 2008; Chaisson
and Pevzner, 2008; Dohm et al., 2007; Jeck et al., 2007; Li, Zhu, et al., 2010; Margulies et al.,
2005b; Simpson et al., 2009; Treangen et al., 2011; Zerbino and Birney, 2008). These tools
mainly rely on two algorithms, and differ mostly in the way they deal with sequencing
errors and inconsistencies and sequence repeats. Since tools utilized today could be either
deprecated or significantly changed in the near future, we will not address the underlying
advantages and disadvantages for each specific tool. We will, however, cover some of the
more general inherent challenges of deep sequencing data assembly and recommended
optimization methods.
Assembly Algorithms - Currently there are two main models for deep sequencing data
assembly, Overlap-Layout-Consensus (OLC) (Myers, 1995) which calculates overlaps by
(computationally expensive) pairwise alignments, and de Bruijn graph-based (DBG) which
creates a shared k-mer dictionary for the assembly process. K is often set by the user and it is
recommended that it be set large enough so that most overlaps are true and do not occur by
chance, and short enough so as to allow overlap between related sequences. Since
comprehensive reviews are available on these algorithms (Miller et al., 2010), we will focus
more on specific algorithm related considerations for tool selection. A recent overview
comparing the performance of a variety of tools for assembly under different conditions
(Zhang et al., 2011), recommended the use of OLC based assemblers (Hernandez et al., 2008;
Margulies et al., 2005b) for small scale (e.g microorganisms) genome assemblies While


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reserving the use of the less computationally demanding DBG based tools for the assembly
of large (eukaryote) genomes (Butler et al., 2008; Li, Zhu, et al., 2010; Simpson et al., 2009;
Zerbino and Birney, 2008). Another consideration is the read size, with OLC being most
appropriate for a limited number of fairly long reads (~100-800 bp) and DBG more suited
for the assembly of millions of short reads (25-100 bp) (Miller et al., 2010). One should note
that DBG based tool's implementation of specific heuristics reduces CPU demand but at the
cost of higher sensitivity to sequencing errors that could result in a much higher memory
requirement. We therefore urge the user to run a more strict quality assessment and
filtration when embarking on DBG based assembly. We also note that some of the assembly
challenges such as identical repeat regions longer than the sequenced reads length, remain
insurmountable by computational and algorithmic improvements and must be alleviated by
technical means such as longer reads or paired-end sequencing (Cahill et al., 2010).
Quality Assessment - An assemblys quality is measured by it's contiguity and cumulative
size and the accuracy of the assembly. The contiguity is assessed using length statistics such
as contig and scaffold maximal and average length, combined total length and N50 (The
length of the smallest contig in the set that contains the fewest (largest) contigs whose
combined length represents at least 50% of the assembly (Miller et al., 2010)). An assembly's
accuracy is more difficult to assess and external data is usually needed to reveal both miss-
assembly (e.g sequences that are inaccurately joined) and per base accuracy (e.g contigs with
nucleotide mismatches). One way to estimate fidelity is by utilizing paired end reads, re-
aligning them against the assembled contigs to reveal discrepancies in insert size which
probably indicate wrong assembly. When there are available reference sequences they
should be utilized for further validation of the assembled contigs, matching sequences and
marking possible mismatches and chimeras (non-related sequences assembled into one
contig). If no reference sequence is available, it has been shown that the sequence of
available closely related organism (e.g comparative assembly (Pop et al., 2004)) could be
utilized for the same purpose and for contig adjacency assessment (Gnerre, et al., 2009;
Husemann and Stoye, 2010; Meader et al., 2010). A crucial aspect of assembly quality
assurance is the sequence quality. Erroneous sequence reads result in higher computer
memory requirements (especially in DBG based tools (Miller et al., 2010)) and either no
assembly output or wrong inaccurate contigs. As part of the assembly related quality
assurance, it is recommended to discard all reads with ambiguous bases (e.g N) and reads
composed entirely of homo-polymer sequences to alleviate this increase in computational
demand (Paszkiewicz and Studholme, 2010). It is also good practice to trim low quality
bases from read edges and of course remove adapters prior to assembly.
Assembly represents one of the more challenging computational tasks at present and it is
further complicated when implemented on deep sequencing data. General considerations
mentioned in this chapter will help the user to both better understand the challenges
inherent in the sequence data and to match a selected tool's underlying implemented
algorithm with the data at hand and the assembly goals. Moreover, assembly quality could
now be better assessed using the aforementioned parameters, such as N50 and fidelity, in
order to compare assembly tools performance for both existing and future software.
4.3 Variant calling
Variant calling refers to the identification of single nucleotide polymorphisms (SNPs),
insertions and deletions (indels), copy number variations (CNVs) and other types of
structural variations (e.g inversions, translocations etc.) in a sequenced sample (Durbin et


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al., 2010). Detection of these variants from deep sequencing data requires in most cases both
a reference genetic sequence to compare the sequence data against (Li, Li, et al., 2009), and a
specialized variant calling software that utilizes probabilistic methods for correctly inferring
variants. The process is complicated by areas of low coverage, sequencing errors,
misalignment caused by either low complexity and repeat regions or adjacent variants and
library preparation biases (e.g PCR duplicates) (Chan, 2009). Variant calling depends on an
efficient combination between an accurate alignment and sophisticated inference of variance
from it. Since alignment optimization was already discussed in a previous section, in this
section our focus will be more on aspects of variant deduction. We will cover the basic
common challenges and difficulties both general and specific for each variant type, Present
leading bioinformatic tools and databases and their contributions to the field and provide
the user with critical considerations and solutions for some of the aforementioned
challenges.
After initial alignment, certain factors can critically alter the results of variant detection. One
should consider them prior to downstream analysis and implement the appropriate
modifications if necessary.
Depth of coverage Previous studies demonstrated positive correlation between variant
calling sensitivity and increased read depth (Krawitz et al., 2010). Depth can be increased by
either reducing the size of the selected or enriched target region, performing a higher
number of sequencing cycles to produce longer reads to cover the target region or simply
assigning more sequencing lanes. Each method has its benefits and drawbacks. For example,
assigning an additional lane to sequence the same sample requires a higher financial
investment but allows better noise filtration and sequencing errors recognition. Targeting a
specific region increases the coverage and sensitivity at the selected segment, but at the cost
of information loss at the areas outside. After the sequencing process is complete, upper and
lower depth thresholds should be applied on the sequencing data before variant calling is
performed. Setting a lower coverage limit removes erroneous mismatches caused by
sequencing errors and thus supported by very few reads (Durbin et al., 2010; Li, Li, et al.,
2009). Although it is recommended on most tools, setting a lower limit has been shown to
reduce sensitivity without increasing specificity in some tools (Goya et al., 2010) and
therefore should be considered in the context of the utilized tool. Setting an upper limit
removes mismatches caused by copy number variations, PCR duplicates introduced by
library preparation (Gomez-Alvarez et al., 2009) and reads mapping to paralogous
sequences. The limit should be set according to the initial coverage and we recommend
setting the limit to ~10 times the average coverage. PCR duplicates should be further
assessed, removed and marked using specialized tools (Li et al., 2009; PICARD) as
mentioned in the pre-analysis processing section.
Mapping quality and Quality recalibration Some reads mapping to under represented
regions in the genome, especially low complexity and repetitive regions will be inaccurately
mapped with a low mapping quality. SNPs derived from these reads have higher chance of
being false-positives (Durbin et al., 2010) and should be more carefully examined, setting a
more strict quality and coverage threshold if possible. As mentioned in the prior section of
alignment considerations, quality recalibration increases the validity of the alignment
qualities so that they better denote the probability of a mismatch between the base and the
reference. Naturally, these re-calibrated qualities improve the efficiency of variant detection
tools that incorporate alignment qualities into their calling algorithms (Koboldt et al., 2009;
Li, Li, et al., 2009; McKenna et al., 2010; Qi, et al., 2010).


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Cross-lane comparison It is good practice, when different-lane same-sample sequences are
available, to compare the amount of SNPs, insertions and deletions detected for each lane. If
one of the lanes has a significantly higher amount of detected variants, it is probable that it
will introduce false-positives to the analysis and exclusion of that lane from downstream
variant calling is recommended. Another possible data validation option is comparison
against a SNP chip if available (Koboldt et al., 2010). Going over each annotated SNP
provides the user with more than a million checkpoints to ascertain both the validity and
fidelity of the sequencing process, and the chromosomal representation (e.g haploid or
diploid).
We will now address a few more variant specific considerations and applications.
4.3.1 Single nucleotide polymorphisms
After aligning deep sequencing reads against a reference genome, SNPs can be naively
inferred from the results by simply denoting each base that is inconsistent between
reference and read as a SNP. This straightforward inference of mismatches results in a
massive amount of alleged SNPs, many of which suffer from some sort of inaccuracy such
as: calling a mismatch in the wrong location, homozygousity and heterozygousity
discrepancies and even calling a mismatch in the correct location but with the wrong base.
Currently most SNP calling tools (Koboldt et al., 2009; Li et al., 2009; 2008; Li, Li, et al.,
2009; McKenna et al., 2010; Qi et al., 2010) apply different probabilistic based
considerations and heuristics such as quality assessment and recalibration, SNP filtration,
local realignment, coverage assessment, prior probability based on known SNPs,
genotype based likelihood and even cancer genomics (Goya et al., 2010) to elucidate SNPs
from alignment results. The user should be familiar with these considerations and be
aware of the tools that apply each when performing SNP calling. We will go over some of
them and discuss their effects and benefits.
Local realignment Current mapping tools align reads independently of the alignment
region context. If a read's beginning or end maps to a region containing an indel, a mismatch
will be called instead of an indel due to alignment scoring considerations. Adding a
secondary, local alignment that considers reads that support the presence of an indel in the
vicinity of either detected SNPs or known SNP sites retrieved from dbSNP (Day, 2010),
results in a significant reduction in false positive SNPs (Durbin et al., 2010; McKenna et al.,
2010). This local realignment is highly recommended prior to SNP analysis and is either
performed inherently in some tools (Qi et al., 2010) or can be specifically performed using
other available tools (McKenna et al., 2010).
Base Alignment Quality Since local realignment is a computationally intensive process
that depends on correctly denoting insertions and deletions, another method for increased
SNP detection accuracy is purposed (Li, 2011). Implementing a per-base alignment quality
recalibration for re-evaluation of misalignment probability using profile hidden markov
models. This quality recalibration can be performed using SAMtools (Li et al., 2009).
Transition / Transversion Ratio (Ti/Tv) The expected ratio between transitions (e.g purine
purine substitutions) and transversions (e.g purine pyrimidine substitutions) can be
elucidated from empirical data retrieved from the 1000 Genomes project Durbin et al., 2010).
This ratio could be utilized as an initial quality assessment standard. Currently the expected
Ti/Tv ratio is ~2.3 for whole-genome sequencing and around 3.3 for whole-exome
sequencing (coding regions only) (DePristo et al., 2011). When detected SNPs demonstrate a
ratio closer to the expected ratio for random substitutions, with transversions twice as


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common as transitions (e.g ~0.5), low quality variant calling or data is implied and quality
thresholds should be reassessed.
dbSNP validation After producing a list of detected SNPs, it is highly recommended to
compare it against dbSNP, the largest repository of SNP data found within the National
Center for Biotechnology Information database. Detected SNPs present in the database are
considered as known, and the ones not found are considered novel (Li and Stockwell, 2010).
The portion of novel SNPs detected in a deep sequencing experiment should range between
1 and 10 percent (DePristo et al., 2011). If this proportion is higher, a high rate of false
positive variants is suggested and we recommended reevaluating the detection process and
possibly implementing a more strict variation inclusion criteria.
4.3.2 Insertions and deletions (Indels)
Indels are the second most common type of polymorphism and the most common structural
variant, in this sub-section we will address only short indels as the next section will deal
with the larger (>1000kb) structural variants. Most indels range between 2-16 bases in length
(Mullaney, et al.,2010) (also referred to as micro-indels) and their frequency has been shown
to vary across the genome with lower rate in conserved and functional regions and an
increased rate in hot spots for genetic variation. The average indel rate is approximately one
indel in 5.1 to 13.2 kb of DNA (Mills et al., 2006). Their presence implicates on the
pathogenesis of disease, gene expression and functionality, viral disease forms identification
and they can be used as genetic markers in natural populations. Indels occur in an estimated
rate that is eight-fold lower than SNPs (Durbin et al., 2010). This rate varies extensively
between sequenced individuals, usually due to variability between mapping and detection
tools. Reads covering an indel are generally more difficult to map since their correct
alignment either involves complex gapped alignment or paired-end sequencing inference.
Optimal indel detection is performed by combining application of an appropriate alignment
software and variant detection tool (Albers et al., 2010; Koboldt et al., 2009; Li et al., 2009;
McKenna et al., 2010; Qi et al., 2010; Kai et al., 2009), and careful adjustment of their
parameters according to the suspected variants. As mentioned before in the alignment
section, it is highly recommended to perform indel calling with alignment tools that
implement gapped alignment (Krawitz et al., 2010; Li and Durbin, 2009; Li et al., 2008;
Novoalign). A few considerations when addressing insertion-deletion detection:
Read length Increasing the read length has been shown to improve the ability to map and
detect insertion related reads. Sequence reads 36 bases long, such as the ones produced by
the Illumina GAIIx, have been shown to be inefficient for detection of insertions longer than
3 bases with a complete inability to detect insertions longer than 7 bases. Hence the length of
the sequenced reads should be considered according to the insertion size range suspicion
and adjusted appropriately. Naturally, when insertion size is expected to surpass the read
length it is impossible to detect them using single-end sequencing. Increasing the read
length has also been shown to improve micro-indel (<10 bases) detection sensitivity without
significantly affecting specificity, demonstrating a more efficient method for increasing
coverage than simply producing more reads.
Paired-end reads Indel detection greatly improves when based on paired end reads deep
sequencing data (Mullaney et al., 2010). Both alignment (Li and Durbin, 2009; Li et al.,2008)
and variant detection tools (Kai Ye et al., 2009) utilize paired-end reads so that one of the
reads is used to pinpoint the pair's loci in the reference while the other read can be subjected
to gapped alignment and indel inference. Furthermore, the insert (e.g the unsequenced gap


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between a read pair) can also be used to deduce the presence of an indel (discussed in the
next section).
4.3.3 Structural variants
Structural variants (Feuk et al., 2006) are defined as genomic alterations that involve
segments of DNA that are larger than 1 kb. They include: (1) Copy number variations
(CNV), which are sections in the DNA with a variable copy number when comparing to a
reference genome. Insertions, deletions and duplications are types of CNVs. (2) Segmental
duplications, several copies of DNA segments that are almost identical (>90%) that can
appear in a variable number of copies, also considered a type of CNV. (3) Inversions,
segments in the DNA that are reversed in orientation. (4) Translocations, an intra or inter
chromosomal location shift in a DNA segment without changing the total DNA content. (5)
Segmental uniparental disomy, where a diploid individual's pair of homologous
chromosomes originated from a single parent. Since current deep sequencing platforms do
not produce reads that span the length of structural variants, utilization of paired end
mapping is necessary for their exact elucidation. The quality of structural variation detection
using deep sequencing can be assessed by the accuracy of break point localization, copy
number count and variation size estimation (Medvedev et al., 2009).
Paired-end mapping (PEM) - Paired-end sequencing refers to the process of sequencing a
cloned DNA fragment on both ends, resulting in two associated sequence reads with an
unsequenced insert between them. The insert length varies between several bases to several
thousands of bases and thus appropriate for the detection process of the aforementioned
large scale structural variants. Structural variants are often detected indirectly through
associated paired-end deep sequencing data patterns (Bashir et al., 2008; Korbel et al., 2009;
Medvedev et al., 2009). Some of these patterns approximate the location of the structural
breakpoints, and some provide an exact localization. For example, the signature of an
insertion or deletion can be easily inferred by comparing the expected read pair distance
according to the reference with the expected insert size, if the reference distance is longer or
shorter than the insert size, the presence of a deletion or insertion can be inferred respectively
but the deduction of the exact location of the indel from these signatures is more difficult.
However, in an anchored split mapping signature, when one read from a pair is perfectly
aligned against the reference and its pair cannot be aligned against its designated reference
location, the unaligned read can be utilized in order to pinpoint the exact location of existing
large deletions or small insertions (Medvedev et al., 2009). SV PEM signatures improve all
aspects of SV detection quality and so PEM is highly recommended for this purpose.
Insert size insert size, set by the size of the DNA fragments introduced by library
preparation can affect the outcome of SV detection. If the experimental goal is to detect as
many structural variants as possible, a larger insert length is suggested. If however, a more
precise localization of the variants is necessary, a shorter insert length is recommended,
though resulting in an overall lower variant discovery sensitivity (e.g if you find the variant,
there is a greater probability of precise localization) (Bashir et al., 2008).
Depth of coverage Coverage can also be utilized for SV elucidation, specifically large scale
deletions and duplications (Yoon et al., 2009). As we expect reads mapping to each region to
follow a Poisson distribution, deviations from the expected coverage suggest the presence of
a duplication or deletion. SV detection benefits from combining increased coverage with
abundance of paired-end reads with a significant increase in specificity (Bashir et al., 2008).
Coverage cannot be utilized however to elucidate the exact location of these SVs, only to


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suggest the expected region in some cases. Coverage biases, as mentioned in previous
sections, can also misconstrue the SV detection process.
Clustering methods After SV signatures have been detected, a calculated inference
process is crucial. Most methods utilize some sort of clustering for all the pairs supporting a
variant, and deduce variant information from that cluster. Since most methods rely on PEM
and coverage for SV detection, they differ mainly on their clustering methods. It is
important to familiarize with these methods since some are more suited for certain variation
types. Since describing each clustering method is beyond the scope of this chapter, we will
only point out certain aspects that are both easy to implement and have significant effects on
detection quality. The standard clustering method (Korbel et al., 2009) utilizes only uniquely
mapped read pairs, and discards the ones mapped to multiple loci. It also utilizes a set
standard deviation limit for the difference between known insert size range and the
observed mapped reference distance. These hard filters reduce the effectiveness of both
homozygousity/heterozygousity inference and small scale variation detection (Medvedev et
al., 2009). Soft (Hormozdiari et al., 2010) and Distribution (K. Chen et al., 2009; McKernan et
al., 2009) based clustering methods consider multiply mapped reads and assigns them
according to their supporting context, thus increasing sensitivity for the presence of small
indels and heterozygousity and should be considered when experimentally relevant.
Validation by assembly It is recommended to combine de novo assembly with structural
variant detection in order to validate detected variants. Once the assembly process is
complete, a search for supporting and conflicting sequence contigs should be performed
(Koboldt et al., 2010).
We note that the field of structural variation deduction from deep sequencing data is still in
its infancy and both false-positive and false-negative rates are far from satisfactory
(Hormozdiari et al., 2009). As both sequencing technology improves, raising coverage and
read length, and the algorithmic utilization of such improvements continues, we expect
greater utilization of deep sequencing for SV detection.
4.3.4 Variant classification
Calling variants using deep sequencing data often results in a multitude of detected
variations, even after strict and effective quality filtration as denoted earlier, deep
sequencing data reveals thousands to millions of different variations (Imelfort et al., 2009).
These variations can result in biological effects through introduction of different amino
acids into protein sequences, early termination of coding sequences and alteration of
regulatory elements and splice sites. A natural step following the variant calling process is
annotating the detected variants and elucidating their effect and biological significance,
separating clinically, scientifically and medically relevant variations from neutral, non
functional ones. In a large list spanning this many variants, manual annotation of each
variant effect is neither feasible or accurate. We will therefore cover currently leading
principles for computational classification and prioritization of detected variants.
Initial prioritization - The first step in variation characterization is basic variation
properties deduction. Variant properties such as it's location, whether in a known coding
sequence, non coding transcript, promoter, splice site etc. Once a variation is localized in a
coding sequence, a subsequent analysis of it's frame effect and whether its synonymous (e.g
changing an amino acid) or non-synonymous should be performed. These basic properties
allow initial prioritization of the variation list, considering that coding sequence non-sense


668
mutations are more likely to be functionally relevant than mutations in an unexpressed
genomic sequence. When dealing with an annotated genome, computational tools should be
utilized for this purpose (Conde et al., 2006; Li and Stockwell, 2010; McLaren et al., 2010;
Yuan et al., 2006). We recommend checking the dbSNP version utilized by chosen
annotation tools and strive to employ the most up-to-date version available so as to increase
the availability of variant annotations.


669
Coding sequence variants - In order to ascertain the most likely phenotype affiliated coding
sequence variation from a given list, current variation profiling methods utilize biochemical
and physical properties of both amino acids and proteins considering both structure
(Ramensky et al., 2002) and function (Bromberg and Rost, 2007; Calabrese et al., 2009) and
utilizing various probability algorithm (Mi, et al., 2007). Possible incorporated characteristics
include: molecular mass, polarity, acidity, basicity, aromaticity, conformational flexibility
and hydrophobicity of amino acids (Ng and Henikoff, 2006) and hydrogen bond breaks,
introduction of a buried polar residue, loss of salt bridge, insertion of proline into -helix,
and the breaking of disulfide bonds in proteins (Wang and Moult, 2001). Some available
tools (Ashkenazy et al., 2010; Kumar et al., 2009; Li et al., 2009) utilize the fact that
functionally crucial amino acids are evolutionary conserved, by employing multiple
sequence alignment based conservation scores in order to prioritize given variations.
Utilizing orthologous sequences for this purpose demonstrates higher efficiency than
incorporation of paralogous, since the latter represents proteins with slight differences in
both sequence and function and is less informative for conservation analysis. It was shown
that conservation degree is in fact the most reliable method for predicting possible
pathogenicity of a missense variant (Flanagan et al., 2010).
For the purpose of both prioritization and functional analysis optimization, we recommend
combining available annotation tools that employ a variety of prioritization features (George
et al., 2008; Lee and Shatkay, 2008). A recent study implemented some of these variation
classification methods on recorded SNPs in a target gene, in order to elucidate possible
cancer causing mutations, reducing the initial number of suspected SNPs from thousands to
less than 30 (Choura and Rebai, 2009). Another study utilized bioinformatic tools to classify
known non synonymous mutations in colon cancer and was able to pinpointed four SNPs
already known as related to increased cancer risk (Doss and Sethumadhavan, 2009).
However, a recent comprehensive review (Karchin, 2009), implemented and compared
leading variant classification tools on three different studies (Doecke et al., 2008; Fatemi et
al., 2008; Van Deerlin et al., 2008) associating both exonic and intronic, novel and known
SNPs with a variety of disease, and demonstrated that a combination of several tools can
possibly result in conflicting annotations and functional effects deduction. Another
comparison (Thusberg et al., 2011), that tested the performance of several of the
aforementioned tools in predicting pathogenicity using test data retrieved from dbSNP,
demonstrated the sensitivity characterizing these tools to range between 0.59 to 0.9, with the
preferred tools for their analysis to be SNPs&GO and MutPred (Calabrese et al., 2009; Li et
al., 2009). Both studies agree that inference of functionality and pathogenicity is not a fully
automatic pathway and educated interpretation of the results must be conducted.
5. Conclusion
Deep-sequencing data analysis is a growing field with many computational challenges. A
normal deep sequencing run outputs a massive amount of data which require complex
computational processing and interpretation. The overflow of available bioinformatic tools
and software for each of the optional analysis steps presents a challenge for the researcher
aiming to evaluate and interpret deep sequencing data. In this chapter we familiarized the
reader with crucial concepts and considerations for preparation, refinement, analysis and
elucidation of valid and accurate conclusions. The field is rapidly evolving both in hardware
and sequencing platform technology and in computational techniques, algorithms, software


670
and tools. It is crucial to understand the various challenges involved in deep sequencing
experiments, and the current available solutions, both in concept and in practice. The
concepts presented in this chapter are aimed towards optimizing deep sequencing
experiments, concentrating on initial steps of data preparation and quality refinement and
covering several possible analysis pathways while denoting some of the currently available
and leading tools, and some of their underlying methods.
The first section of this chapter, introduced deep sequencing technology's available
platforms in regards to their advantages and limitations, emphasizing that although they are
all considered high throughput sequencing platforms, they present different capabilities and
proficiencies. When a choice between platforms is available, one can improve data retrieval
and validity simply by matching the most appropriate platform with the specific
experimental needs.
The second section covered the concept of deep sequencing data quality control. Using
bioinformatic tools, based on both empirical and probabilistic deduction, sequencing
derived errors can be reduced which otherwise would be incorporated into downstream
analysis. We described current quality scales, with methods for their assessment and their
relevance for improved data retrieval. Employment of these quality control and assurance
methods can assist in uncovering biased sequencing lanes and recurring errors and
contaminants that could significantly alter deep sequencing results. We therefore strongly
urge users to utilize them prior to any following experimental evaluation, making their
incorporation a standard in deep sequencing experiments.
The third and subsequent sections covered specific and very common analysis pathways:
alignment, assembly and variant calling. The chapter introduced basic challenges faced in
each type of analysis, their current limitations and considerations pivotal for preferential
experimental planning. A description of each challenge was accompanied by delineation of
current methods, tools and solutions when available. Familiarized with these challenges, the
user can now conduct better analytic decisions and employ the most appropriate tools and
techniques. Understanding the exact edge of each analytic pathway can help the user to
perform their deep sequencing experiments in the most effective manner employing both
current and future software for optimal variant calling.
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30
Whole Genome Annotation: In Silico Analysis
Vasco Azevedo et al.*
Federal University of Minas Gerais (UFMG) and Federal University of Par (UFPA),
Brazil
1. Introduction
After a genome is assembled, the next step is genomic annotation, which can generate data
that will allow various types of research of the model organism. Complete DNA sequences
of the organism are then mapped in areas pertinent to the research objectives. In this
chapter, we explore relevant ongoing research on genes and consider the gene as a basic
mapping unit. Gene prediction is the first hurdle we come across to begin the extensive and
intensive work demonstrated in first item, which deals with assembly of the genome. Gene
prediction can be made with computational techniques for recognizing gene sequences,
including stop codons and the initial portions of nucleotide sequences; it involves empirical
rules concerning minimum coding sequences (CDS's) and is limited due to overlapping
sequences coding forward and reverse.
Finishing gene prediction step by a computer initiates the functional annotation stage.
Functional annotation, item 3, can be done initially by computer, using similarity in
sequence alignment. However, no software is capable of generating a functional
annotation without many false positive results, since conserved protein domains with
varied functions make gene sequence alignment difficult. In this case, after automatic
annotation, the predicted genes need to be revised manually. In manual curation, item 4,
an expert can more accurately locate frameshifts in the DNA strand. Depending on the
number of errors found, genomic annotation may be postponed, requiring a return to the
previous stage of genome assembly. In manual curation, the principal contributions are
usually correction of the start codon position, gene name, gene product and, finally,
identification of frameshifts.
When functional annotation is completed, the genome should subsequently be submitted. It
occurs after the assembly and annotation steps making the data generated available in
public-access databanks. Submission is a pre-requisite for publication in scientific journals.
Another advantage of genome publication in public-access sites is that it permits use of
various genome analysis tools. For example, searches for genomic plasticity, pangenomic
study, exported antigens and evaluation of innate and adaptive immune responses. The
pangenome approach, item 5, concepts of species can be used as a filter for targeting
candidates for vaccines, diagnostic kits and drug development. For drug development, the

*
Vinicius Abreu, Sintia Almeida, Anderson Santos, Siomar Soares, Amjad Ali, Anne Pinto, Aryane
Magalhes, Eudes Barbosa, Rommel Ramos, Louise Cerdeira, Adriana Carneiro, Paula Schneider, Artur
Silva and Anderson Miyoshi
Federal University of Minas Gerais (UFMG) and Federal University of Par (UFPA), Brazil


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core set of proteins is a more likely source of useful information, for developing both
vaccines and diagnostic materials for a unique pangenome set of a species of interest.
Genomic plasticity, item 6, is the dynamic property of genomes, involving DNA gains,
losses, and rearrangement; it allows bacteria to adapt to new hosts and environments. There
are several mechanisms that can drive these changes, including point mutations, gene
conversions, rearrangements (inversion or translocation), deletions and DNA insertions
from other organisms (through plasmids, bacteriophages, transposons, insertion elements
and genomic islands). Gene acquisition and loss by all these mechanisms influences
bacterial lifestyles and physiological versatility. Analyses of HGT regions in silico has
become feasible due to the introduction of nextgeneration sequencing technologies, which
allows sequencing of prokaryotic genomes at a faster rate than the earlier Sanger method
and at a considerably lower operational cost. Consequently, the number of complete
genome sequences available for analysis has grown and continues to grow rapidly.
In post-genomics, study of Reverse Vaccinology (RV), item 7, can provide predictions of the
sub cellular locations of an entire predicted proteome. Additionally, these previous
annotations, prediction of peptides with high affinity for class I and II MHC proteins is
another in silico analysis that increases the probability of selecting antigens that can
promote immune responses in organisms infected by a pathogen. The field of research
referred to as immunoinformatics, item 8, is giving us the opportunity to analyze antigens
with greater selectivity and increase the likelihood of developing a successful vaccine.
2. Gene prediction
The development of modern sequencing technology has resulted in an exponential increase
in the number of available genome sequences. To illustrate, in 1997 there were 10 complete
genome sequences of bacteria available in the NCBI (Lukashin & Borodovsky, 1998); by
2011, this number had sharply increased to 1,538 http://www.ncbi.nlm.nih.gov/
genomes/lproks.cgi. This enormous increase in the quantity of available information
stimulated the development of tools for gene prediction. The development of these tools is a
tremendous challenge, and it is a major contribution of Bioinformatics to the field of genomics.
2.1 Gene prediction strategies
Gene prediction programs can be divided into two categories: an empirical category, which
relies on sequence similarity; and ab initio, which uses signal and content sensors. Empirical
gene predictors search for similarity in the genome; they predict genes based on homologies
with known databases, such as genomic DNA, cDNA, dbEST and proteins. This approach
facilitates the identification of wellconserved exons. Ab initio gene finders use sequence
information of signal and content sensors. Usually, these programs are based on Hidden
Markov Models. Ab initio can be organized into categories based on the number of genome
sequences used in gene analysis; it includes single, dual and multiplegenome predictors.
Integrated approaches couple the extrinsic methodology of empirical genefinders and
intrinsic ab initio prediction. This technique significantly improves gene prediction
protocols (Allen et al., 2004).
2.2 Eukaryotes
The complexity of the challenge faced by Bioinformatics is only completely understood
when we look at the complexity of the eukaryotic genome. Within genomes, genes are not


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organized in a continuous cluster. Instead, the coding regions (exons) are often widely
interspersed with noncoding intervening sequences (introns). Furthermore, in many cases
the intronic region is much larger than the exonic region. These lowdensity coding
sequences are evident in the human genome, in which only approximately 3% of the DNA
generates proteins. The exon and intron issue can be compared to trying to read a non
continuous article in a journal. In an analogy, one must first identify in which part of the
journal (genome) the article (gene) of interest is; then, as the DNA sequences are read, it is
necessary to identify which part is informative (exon) and which part contains random
information (intron). Also, genes can be altered by alternative splicing, which is a process
that generates multiple protein sequences from the same gene sequence template
(Schellenberg et al., 2008).
Gene prediction methodology for eukaryotes involves two distinct aspects; the first focuses
on the information utilized for gene recognition, basically recognizing signal functions in the
DNA strand; the second uses algorithms implemented by prediction programs for accurate
prediction of gene structure and organization. The signal function search can be divided
into two mechanisms utilized for locating genes. One classifies the content of the DNA
strand and the other searches for functional signals in the genome:
(i) The content sensor classifies the DNA regions into coding and non-coding segments
(introns, intergenic regions and untranslated regions). This mechanism involves two
approaches, intrinsic and extrinsic. The extrinsic approach relies on the assumption that
coding regions are evolutionarily more conserved than noncoding regions. Consequently,
this methodology employs local alignment tools, like BLAST (Johnson et al., 2008) ; this
makes it possible to make comparisons within the genome and between closely-related
species. However, one important flaw in this approach involves the necessity of identifying
homologies within the database in order to extract results. If none is found, this
methodology is unable to determine if a region "codes" for a protein (Sleator, 2010). (ii) The
functional sensor approach searches the genome for consensus sequences. Consensus
sequences are extracted from multiple alignments of functionally-related documented
sequences. The functional signals involve transcription, translation and splice sites.
Transcriptional signals includes the CAP signal at the transcriptional start site and the
polyadenylation signal located 20 to 30 bp downstream of the coding region. Another
important signal to identify is the translation initiation site, although this feature has
limitations due to a lack of knowledge concerning initiation sites in eukaryotes (Math et al.,
2002).
2.3 Prokaryotes
Unlike eukaryotes, the archaeal, bacterial and virus genomes are highly gene-dense. The
protein coding regions usually represent more than 90% of the genome. Therefore the
accuracy of gene predictors depends primarily on determining which of the six frames
contains the real gene. The simplest approach in gene prediction is to look for Open Reading
Frames (ORFs). An ORF is a DNA sequence that initiates at a start codon and ends at a stop
codon, with no other intervening stop codon. One way to locate genes is to look for ORFs
with the mean size of proteins (roughly 900 base pairs) (Allen et al., 2004). Therefore, long
ORFs indicate possible genes, although this methodology fails to predict small genes.
The major problem in simply applying this technique is the possibility of ORF overlap in the
different DNA strains. This approach must be used along with guidelines to avoid


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overlapping, choosing the more likely candidates. Also, numerous false positives are found
in non-coding regions. Due to the high gene density, it is difficult to confidently state that
any gene predicted in a noncoding region is false. This problem can be minimized by
searching for homologies in closelyrelated organisms. If we do not find a conserved
sequence in related species, it is assumed that the prediction (of a gene) is false.
Another problem faced by prediction programs in prokaryotes is how to determine the start
codon of a sequence. The first initiation site in a sequence is not necessarily the true one. To
solve this problem, programs can employ ribosome binding sites (RBS), which provide a
strong signal, indicating the position of the true start site. In conclusion, there is a drop in
prediction accuracy in highGCcontent genomes. Rich GC genomes contain fewer stop
codons and more spurious ORFs. These false ORFs are often chosen by prediction programs
instead of the real ones in the same DNA region. Additionally, the longer ORFs in GCrich
genomes contain more potential start codons, leading to a drop in the accuracy of
translation initiation site prediction (Hyatt et al., 2010).
2.4 Tools
2.4.1 Glimmer
The first version of Glimmer (Gene Locator and Interpolated Markov ModelER) was
released in 1998 ; the 3.02 version was released in 2006. Glimmer is a system for finding
genes in microbial DNA, especially the genomes of bacteria, archaea, and viruses. Glimmer
uses interpolated Markov models (IMMs) to identify coding regions and distinguish them
from noncoding DNA. Glimmer was the primary microbial gene finder used at The Institute
for Genomic Research (TIGR), where it was first developed, and it has been used to annotate
the complete genomes of over 100 bacterial species from TIGR and other labs. Like other
gene prediction programs, Glimmer can be installed and run locally and has a web-based
platform (Salzberg et al., 1998). All one needs for online gene prediction of a genome is the
fasta version of the sequence and access to the site:
http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi.
2.4.2 FgenesB
FgenesB is a package developed by Softberry Inc. for automatic annotation of bacterial
genomes. The gene prediction algorithm is based on Markov chain models of coding
regions and translation and termination sites. The package includes options to work on
sets of sequences, such as scaffolds of bacterial genomes or short sequencing reads
extracted from bacterial communities. For community sequence annotation, it includes
ABsplit program, which separates archebacterial and eubacterial sequences. FGENESB
was used in the first published bacterial community annotation project (Tyson et al.,
2004).
2.4.3 Prodigal
Prodigal (Prokaryotic Dynamic Programming Genefinding Algorithm) is a microbial
(bacterial and archaeal) gene finding program developed at Oak Ridge National Laboratory
and the University of Tennessee. Prodigal focuses specifically on three goals: improved gene
structure prediction, improved translation initiation site recognition, and reduced false
positives (Hyatt et al., 2010). The source code is freely available under the General Public
License and the program can be accessed at http://compbio.ornl.gov/prodigal/.


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2.4.4 GeneMarkTM
GeneMark is a public access program for gene prediction in eukaryotes. It is a family of gene
prediction programs developed at Georgia Institute of Technology, Atlanta, Georgia, USA.
GeneMark can operate in two ways: the first one is online, where one can make predictions,
using for comparison one of the many available models; the second option is for novel
genomes, in this way one can install and run the program locally. The webbased version of
GeneMark is available at http://exon.biology.gatech.edu/.
For gene prediction in eukaryotes, GeneMark combines two programs, GeneMarkE* and
GeneMark.hmmE. The GeneMark-E program determines the protein-coding potential of a
DNA sequence (within a sliding window) by using species-specific parameters of the
Markov models of coding and non-coding regions. This approach allows delineating local
variations with coding potential. The GeneMark graph shows details of the protein-coding
potential distribution along a sequence, while the GeneMark.hmm-E program predicts
genes and intergenic regions in a sequence as a whole. The Hidden Markov models take
advantage of the "grammar" of gene organization. The GeneMark.hmm programs identify
the most likely parse of the whole DNA sequence into protein coding genes (with possible
introns) and intergenic regions.
The statistical model employed in the GeneMark.hmm algorithm is a hidden Markov model.
It includes hidden states for initial, internal and terminal exons, introns, intergenic regions
and single exon genes. It also includes hidden states for start site (initiation site), stop site
(termination site), and donor and acceptor splice sites. The protein-coding states (initial,
internal, terminal exons and single exon genes) emit nucleotide sequences modeled by
inhomogeneous 3periodic fifthorder Markov chains. The non-coding states (intron and
intergenic regions) emit sequences modeled by homogeneous Markov chains (Lukashin &
Borodovsky, 1998).
3. Automated functional annotation
Automated functional annotation of genomes can be quite efficient because it is a
computational process based on the alignment of ORF sequences of the organism with
sequences from various other organisms (Kislyuk et al., 2010). Public domain databases
contain full annotations of thousands of prokaryotic organisms (Benson et al., 2008).
Automatic functional annotation takes advantage of knowledge concerning ORFs of
homologous organisms, saving considerable time in manual curation (Li et al., 2010).
However, care must be taken with fully automated functional annotation, since similarity of
sequences can easily incur false positives (Lorenzi et al., 2010). In this section we discuss the
advantages and dangers of using fully-automated functional annotation, and we explore
some features of tools and services for this purpose.
3.1 Massive sequence alignments must be planned
Algorithms for alignment of biological sequences are intensively used in automatic
functional annotation (Aparicio et al., 2006; Meyer et al., 2003). Alignments of ORFs from a
newly assembled genome with counterpart ORFs can provide the first hints about the new
genome. For an organism with about 2,000 ORFs, analysis of similar sequences against a
database of non-redundant (NR) proteins from NCBI can consume several processing hours.
For example, assuming that this analysis is done on a computer isolated from the internet,
hardware with 24 Gb RAM and eight processors, totaling 24 GHz CPU, this task will
consume approximately eight hours of processing time.


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Though it is a completely automated computer process, the user has considerable
responsibility to set conditions to be utilized in the computation in order to obtain good
quality data. These conditions define the quality criteria that best fit the type of organism,
for example, the cutoff value for a significant alignment with sequences of other organisms
in the NCBI, the number of homologous sequences to be returned as a result and the file
format of the output alignment. An additional parameter is required if the sequence search
(query) and the targeted search sequences (subject) are in different formats (nucleotides
versus amino acids). This parameter determines the most adequate table for translation of
codons of the organism in question so that the alignment algorithm of sequences is able to
interpret the correspondence between the query sequences and the subject. The number of
parameters of an algorithm for aligning sequences can be quite large, justifying training
with a heavy workload for optimal utilization. Our objective here is not to explore possible
situations, but to alert users that the results of these algorithms can improve these
alignments by reading the manual algorithm and consequently adjusting it to a particular
situation concerning a query organism or subject. Thus, when beginning a massive
alignment sequences project involving a novel genome, with an analysis that will take hours
and create high expectations, it is advisable to use not just the basic configurations in these
alignment algorithms. It would be useful to take time to weigh and incorporate options that
will determine the success or failure of these alignments.
3.2 Knowledge reapplication and time saving
There has been significant growth in the number of DNA sequences available in public
databases, because of new genome sequencing technologies, which have made it simpler,
more efficient and cheaper to obtain complete genomes (Zhao & Grant, 2010). Fully
assembled and annotated genomes of various forms of bacterial life are available to facilitate
the processing and inclusion of a newly assembled genome. This wide range of genomes
provides the opportunity for new research into large-scale SNPs, DNA methylation and
mRNA expression profiles, and resequencing data (Datta et al., 2010). It also allows
comparison of annotations from different research groups working with different
organisms, some of which may be homologous to a newly-sequenced genome. Just as one
can take advantage of knowledge about the function of genes from different organisms, it is
also advisable to use the personal knowledge of a researcher on a specific organism in order
to accelerate the process of automatic annotation. Based on evidence about a high degree of
evolutionary proximity between a newly-assembled genome and a particular organism
homolog that already has a fully-assembled and annotated genome, we can choose to use
only the annotation of such an organism as a resource for a first automatic annotation.
The problems a researcher would normally encounter when utilizing annotations from
various genomes could be resolved by comparison with the annotation of a homologous
organism. This situation is common when one examines the pangenome of a species, as it is
expected that most of the coding sequences of different strains of bacteria are not very
different (Trost et al., 2010). In this case, it appears to be advantageous to identify a small set
of target organisms (subject) in a sequence similarity search, with the objective of providing
a first genome annotation (query); this may even be a set with only one organism.
3.3 Error propagation: Automated versus manual annotation
It is important to bear in mind that the GenBank is not a fully curated database (Benson et
al., 2008); many genomes may have been deposited only as automatic annotations. With


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current technology, it is not possible to dispense with manual curation of an automatic
annotation, or even experimental evidence concerning gene prediction and annotation based
on sequence similarities (Poptsova & Gogarten, 2010). Although it is not normally feasible to
initially include experimental verification of gene prediction, it seems reasonable to take
advantage of expert human annotation of genomes to help determine the outcome of
automatic annotation. Assuming one is working on the pangenome of an organism, such a
measure can not only reduce false positives in comparisons of sequence similarities, but also
determination of homologous genomes based on a particular annotation. During automatic
annotation, a measure that has the potential to minimize error propagation would be
allocating different weights for the results of sequence similarity to genes from organisms
for which there is evidence of expert manual curation.
3.4 Tools
The following are some tools for automatic annotation of entire genomes, with brief
descriptions of their core functionality and instructions on how to use them.
3.4.1 GenDB
One of the reasons that GenDB is included among a select set of tools for automatic
annotation of genomes is the fact that it was developed for the web platform (Meyer et al.,
2003). Geographically dispersed research groups can benefit from web interfaces using
standard tools and a centralized database. Version 2.4 of GenDB has three modules: core,
web, and gui. The core module has programs written in perl that allow creation of an
annotation project, importation of data in fasta / EMBL format, execution pipeline
automatic annotation, display of circular genomic maps, data export and annotation project
deletion. Implementation of the programs in the module allows a team of curators to work
on the web and edit diverse features of various genes. The gui module has editing features
that are more sophisticated than those of the web module, allowing execution of tasks
performed by the core module, but with a graphical interface. The GenDB program
performs sequence alignments using the program Blast (Altschul et al., 1997) and allows
incorporation of predictions of conserved domains of protein families based on InterPro-
Scan (Hunter et al., 2009),as well as transmembrane domains based on TMHMM (Krogh et
al., 2001), and indications of export to the extracellular medium through SignalP (Bendtsen
et al., 2004).
3.4.2 BLAST2GO (B2G)
This tool was designed as an interface for Gene Ontology (GO); additional features have
transformed it into a more comprehensive annotation platform (Aparicio et al., 2006). The
program menus include various steps initiating annotation, with an automatic alignment of
genome sequences against a protein-based non-redundant (NR) NCBI database, through
prediction of conserved domains (InterProScan), GO annotation ratings against the
enzymatic English Enzymatic Code (EC) and subsequent visualization of molecular
interactions in a genome by means of maps in the format of the Kyoto Encyclopedia of
Genes and Genomes (KEEGO). Being a visually oriented tool, it has graphical tools to help
analyze the vast amount of data generated in the predictions. A user of B2G does not
necessarily have to perform all the steps of analysis that are offered, but in order to advance
to the next phase of analysis it is imperative that the previous phase be performed


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beforehand. Processing of an entire genome with approximately two thousand ORFs can take
several days, as the first step is always sequence alignment against the NCBI NR base.
Fortunately, B2G is designed to be a modular analysis tool. If a B2G user has computational
resources that are more efficient than the shared resources on the public server, the user can
perform alignment of sequences on his own hardware to generate an output in HTML format
and continue the alignment processes following annotation with B2G. Should the user be
dissatisfied with the efficiency of processes of annotating GO terms of the server's common
B2G, there is a version of B2G than he can run separately with his superior hardware. The
results generated in the offline mode can be uploaded to the online tool to continue the
review process using a variety of tools, including statistical comparisons between two
genomes. B2G was developed with Sun Java technology, which can be run on any operating
system; however, the B2G offline module is designed to run on the Linux platform.
3.4.3 CpDB relational schema: a practical example
This tutorial has approximately 100 steps, including software installation and configuration,
edition of files by Linux commands or through interfaces with biological sequence
manipulation programs. The tutorial presumes that the programs Artemis, Java (Sun) and
Blast version 2.2.20 or previous were locally installed. Many editions of files are made with
the "sed" program of Linux, which is included in most Linux versions. All of the steps in this
manual can be automated in order to develop an automatic pipeline for annotation,
allowing the Corynebacterium pseudotuberculosis DataBase (CpDB), a relational database
schema and tools for bacterial genomes annotation and pos-genome research, to become
another web-based automatic annotation environment. For now, this tutorial has an
instructional character, to help make a student aware of the necessities and difficulties
involved in the process of automatic annotation of genomes. In order to obtain the tutorial
files, type the following command in Linux, Ubuntu 10.10 or later:
svn checkout svn://150.164.37.20/genomes/autoannotation --username=student --
password=bioinfo
After finalizing the verification of all of the files, this tutorial continues in the document
"Tutorial.pdf", which will be in the folder "autoannotation".
4. Manual curation
Genome annotation is a process that consists of adding analyses and biological
interpretations to DNA sequence information. This process can be divided (Stein, 2001), into
three main categories: annotation of nucleotides, proteins and processes. Annotation of
nucleotides can be done when there is information about the complete genome (or DNA
segments) of an organism. It involves looking for the physical location (position on the
chromosome) of each part of the sequence and discovering the location of the genes, RNAs,
repeat elements, etc. In the annotation of proteins, which is done when there is information
about the genes (obtained by genome or cDNA sequencing) of an organism, there is a search
for gene function. Besides general predictions about gene and protein function, other
information can be found in an annotation, such as biochemical and structural properties of
a protein, prediction of operons, gene ontology, evolutionary relationships and metabolic
cycles (Stothard & Wishart, 2006). Consequently, functional annotation or manual curation
is a fundamental part of the process of assembling and annotating a genome, in which the
curator is the person responsible for validating the elements. In manual curation, all of the


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predicted genes will be validated and their products named (Stein, 2001). A more detailed
description of the gene or gene family product is obtained through similarity analyses using
protein data banks that contain well-characterized and conserved proteins (Overbeek et al.,
2005).
4.1 Technical terms used in manual annotation
In functional annotation done with Artemis, several fields should be filled out to increase
knowledge about particular genome elements. It is necessary to use annotation terms, which
involve an official nomenclature developed for this purpose. Some of these terms and
respective examples are given below: "LOCUS-TAG" is the term used to identify all of the
genome elements, except for the feature "misc". Generally, one uses an abbreviation to
identify the particular species, followed by an underline (_) and numbers, for example:
Cp1002_0001 (Corynebacterium pseudotuberculosis, strain 1002). For tRNAs, the nomenclature
is the abbreviation, followed by underline, a "t" and numbers, with a specific count, which is
not included in the total CDS count, among others; for example: Cp1002_t001. For rRNAs,
the nomenclature is the symbol followed by underline, an "r" and numbers, with specific
counts, not included in the total CDS count; for example: Cp1002_r001. "PROTEIN_ID" is
used to designate all of the elements of the genome, except for the feature "misc". It is a
standardized form for NCBI to identify e proteins; for example: gnl|gbufpa|Cp1002_0001.
"GENE" is one of the most important topics to be informed in manual annotation, indicating
the gene symbol of the protein; fore example: pld. The field "SIMILARITY" corresponds to
information obtained from the best similarity search result BLASTp. Various types of
information should be entered into this field, such as similarity among organisms, size of the
amino-acids sequence analyzed, e-value and also the percentage identity between its own
protein and the protein found in the data bank; for example: similar to Corynebacterium
pseudotuberculosis 1002, hypothetical protein Cp1002_00047 (345 aa), evalue: 0.0, 98% ID in
344 aa. In "PRODUCT", there is a description of the gene product, for which similarity was
found in the public domain data bank; for example: Phospholipase D. The tag "PSEUDO"
should be added whenever a protein presents one or various breaks, due to insertion of a
premature stop codon. These are the famous proteins that have frameshifts or probable
pseudogenes. Consequently, the manual annotation window has this pattern:

/gene="dnaA"
/product="Chromosomal replication initiation protein"
/locus_tag="Cp1002_0001"
/protein_id="gnl|ufmg|Cp1002_0001"
/colour=3
/similarity="Similar to Corynebacterium pseudotuberculosis FRC41,
Chromosomal replication initiation protein (603 aa), e value: 0.0, 98% id in 599s aa"
4.2 Steps for manual curation
Manual curation is a very complex task and is subject to errors for various reasons. One of
these is a lack of padronization in the interpretation of BLAST results. Another problem is
propagation of errors, which involves prediction of protein function based on proteins that
were also predicted but could have imprecise or even incorrect annotation (Gilks et al.,
2002). For these reasons, some criteria are suggested in order to obtain reliable functional


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annotation. The fundamental step for doing this well is mining data obtained from
similarity analyses of BLASTp data banks. It is recommended to give greater value to
annotation of proteins of individuals of the same species or of species that are
phylogenetically close to the organism under study, the protein of which one wants to infer
the function of, decreasing in this way the possibility of annotation errors. Another
parameter is to observe if there is any consensus among the first 10 hits (the same protein is
identified among various). In this case, even if the best hit is not identified as such, it is
preferable to identify the sequence as similar to that of an organism that appears various
times in the BLASTp results and is within the consensus. In cases where there is no
consensus or when the e-value of the best hit (first BLAST result and which corresponds to
the best alignment within the data bank that is being researched) is significantly larger than
that of the following sequences, it is preferable to transfer the annotation of the best hit
(Prosdocimi, 2003), or if necessary, in cases of non-significant alignments, always also run a
similarity search at the nucleotide level (BLASTn). Other criteria are also analyzed, such as
percentage identity between the sequence being analyzed and the sequence in the data
bank, score value and e-value, as well as pair-by-pair alignment evaluation. This evaluation
consists of checking the texture of the alignment (evaluating the number of gaps, size of the
gaps, and the number of conserved substitutions of amino acids). If doubts remain, research
of domain data banks and protein classification are also commonly utilized.
4.3 Frame shifts (Pseudogenes)
Comparisons between non-coding regions of genomes of various prokaryotic species has
aided in the identification and characterization of genome segments with regulatory roles
(Pareja et al., 2006), contributing to the elucidation of genetic circuits of transcriptional
regulation. These non-coding regions, known as pseudogenes, are DNA sequences that are
highly similar to functional genes but do not express a functional protein, probably because
of deleterious mutations. These degraded genes contain one or more inactivating mutations,
such as a nonsense mutation that introduces a premature stop codon, resulting in an
incomplete protein and a later change in the open reading frame (Lerat & Ochman, 2005).
When found in the genome, the break region is checked with Artemis, and the quality of the
bases in that region is also evaluated. Whenever possible, addition or removal of erroneous
bases can restore the reading frame. If there is no data that justifies addition or removal of
bases, the genes should be classified as pseudogenes (tag /pseudo).
4.4 Tools
4.4.1 Artemis
The program Artemis, (Berriman & Rutherford, 2003), available for download at
http://www.sanger.ac.uk/Software/Artemis is a freely-distributed algorithm developed
for visualization of genomes and for annotation and manual curation. Artemis allows the
curator to visualize various characteristics of the genome sequences, such as: product coded
by the predicted gene; presence of tRNAs and rRNAs; search for protein and nucleotide
similarity in biological data banks; visualization of probable domains and conserved protein
families; visualization of GC / AT content, and misplaced codon use; and various other
functions. These data can be visualized in the six phases of translating DNA reads into
proteins (Rutherford et al., 2000). Also, the program provides a visualization of BLAST visits
between two complete genome sequences, allowing rapid analysis of the degree of synteny


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(conservation at the level of genes), the main genomic rearrangements and integration of
new genomic islands (Field et al., 2005). This algorithm is written in the Java language and is
available for the following operating systems: UNIX, Macintosh and Windows. Artemis is
capable of processing data in the formats EMBL and GENBANK, or even sequences in the
format FASTA.
4.5 Sequence similarity searches
4.5.1 BLAST (Basic Local Alignment Search Tool)
BLAST (Altschul et al., 1990) is a tool that is widely used for the characterization of products
coded by genes that are identified by gene prediction. It is able to identify a great majority of
the alignments that attend the desired criteria, with a significant gain in performance (Gibas
& Jambeck, 2001). This program is available on the NCBI - National Center for
Biotechnology Information site http://www.ncbi.nlm.nih.gov (Stein, 2001), which is
considered the central databank for genome information. As shown in the figure, BLAST has
programs for alignment of protein and nucleotide sequences, among others, according to the
needs of the work that is to be undertaken:

Program Entry sequence Type of sequence target
BLASTp Protein Protein
BLASTn Nucleotide Nucleotide
BLASTx Translated nucleotide Protein
TBLASTn Protein Translated nucleotide
TBLASTx Translated nucleotide Translated nucleotide
Table 1. Types of BLAST NCBI programs.
Through this type of algorithm, we can compare any DNA sequence or protein (query) with
all of the genome sequences in the public domain (subject) (Altschul et al., 1997). It is
important to note that the program BLAST does not try to make a comparison of the full
extension of the molecules that are being compared, but rather it identifies in the data bank
a sequence that is sufficiently similar to that of the sequence that is being studied.
4.5.2 Interpreting blast results
In the manual annotation of genomes, analysis of BLAST parameters, such as the number of
points obtained (score), gap opening/extension penalties, number of expected alignments in
the case of scores equal to or superior to the alignment that is being investigated
(expectation value), and the normalized score (bitscore), are indispensible for the
interpretation of the results. The smaller the value of "E", the smaller the chance of such a
comparison being found merely by chance, consequently inferring a greater homology
between the sequence being investigated and the data base (Baxevanis & Ouellette, 2001).
Among the sequences with identity above 50%, a general approach is to characterize the
function of the known sequence and transfer this annotation to the new sequence. Though
annotation transfer is a common practice, a high rate of error has been reported when this is
done without due caution (Liberman, 2004). Based on this principle, we consider that for
sequences with identity above 80%, a simple alignment or a comparison with a protein that
has been experimentally characterized using BLAST can be sufficient to infer function, as
long as the pair being compared has similar lengths and align end to end without large


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deletions or insertions. For pairs with identity in the range of 5080%, the general approach
for attributing function includes evaluation of databanks with homologous protein and
protein domain families.
4.5.3 PFAM
Proteins generally are composed of one or more functional regions, or domains. Different
combinations of domains result in the large variety of proteins found in nature.
Identification of the domains that are found in proteins can, therefore, provide insight about
protein function (Sanger Institute, 2009). In sequences with an identity of less than 70%,
without end to end similarity, the approach that is used is to evaluate the protein domains
through a search of the Pfam database, which gives very extensive coverage (Mazumder &
Vasudevan, 2008). The Pfam database is accessible via the Web http://pfam.sanger.ac.uk
and is available in various formats for download. This databank is contains two
complementary groupings; PfamA is composed of highquality protein domains that have
been manually verified, while PfamB contains data that has been generated automatically
from the ProDom databank (Finn et al., 2010). PfamB is generally lower in quality, though
it can suggest new domains that can be added to the manual annotation, if they are not
available in PfamA. Basically, in Pfam, the sequences that are in full alignment are
identified through a search for a hidden profile using the algorithm Hidden Markov Model
(HMM), which is later generated using the software HMMER, based on the UniProt
database (UniProt, 2007). These HMMs are statistical models that capture specific
information about how much each alignment column is conserved and indicates the
residuals in this evaluation.
5. Genomics
A genome is the complete set of DNA sequences of a living organism; it consists of coding
and noncoding sequences. Genomics is a discipline of genetics that deals with genomes or
DNA sequences. Simply put, genomics is the study of genomes. Computational genomics
derives knowledge from genome sequences and related data, including both DNA and RNA
sequences as well as experimental data. Computational biology mainly deals with whole
genome analysis to understand the DNA mechanisms and molecular biology of a species.
As biological datasets are extremely large, computational biology has become an important
part of modern biology.
5.1 Pangenomics
The efficient and low cost sequencing technologies that are currently available provide
complete genome sequences of pathogenic, industrially useful, and other economically-
important organisms. Genome sequences, and information that is coded in these sequences,
can help identify pathogenicity and other important genes.
Complete genomic sequences of various strains of a species are important to help us
understand pathogenesis mechanisms and to determine how genetic variability affects
pathogenesis; it would be difficult to extract such useful information from a single genome
(Lefbure & Stanhope, 2007).
A pangenome consists of a "core genome", which contains the gene or sequences present in
all strains. In other words, genes that are found in all the genomes in a species of bacteria are


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called the core genome. A "dispensable genome or accessory genome" consists of genome
sequences present in more than two strains but are not part of the core genome. "Unique
genomic sequences" or "unique genes" are strain-specific genes. These genes are limited to
single strain. The pangenome is important for identification and for designing effective
vaccines and drug targets (Mira et al., 2010).
There are many web tools and softwares available to manage and efficiently extract data
from genomes of various strains of the same species. These tools recognize the accession
numbers allotted to complete genomes submitted to NCBI and to other databanks. Online
tools developed by the Computational Genomics group of Bielefeld University, Germany,
EDGAR "Efficient Database framework for comparative Genome Analyses using BLAST
score Ratios" http://edgar.cebitec.uni-bielefeld.de are efficient web tools to determine the
core genome, along with dispensable and unique genes in the form of colored graphs and
tables (Blom et al., 2009)
For example, we analyzed the core genome, dispensable genes and unique genes, using
"EDGAR", of three different Corynebacterium pseudotuberculosis strains, C. pseudotuberculosis
CpI19, C. pseudotuberculosis Cp1002 and C. pseudotuberculosis CpC231.
This core genome consists of 1,862 genes, with 48 dispensable genes between CpI19 and
Cp1002, 52 dispensable genes between Cp-I19 and CpC231, and 103 dispensable genes
between Cp1002 and CpC231. There were 208, 46 and 36 unique genes in strains Cp-I19,
Cp1002 and CpC231, respectively.
6. Genome plasticity
The high degree of adaptability of bacteria to a wide range of environments and hosts is
long known to be influenced by genome plasticity, a dynamic property that involves DNA
gain, loss and rearrangement (Maurelli et al., 1998). Various mechanisms can drive these
changes, including point mutations, gene conversions, rearrangements (inversion or
translocation), deletions and DNA insertions from other organisms (plasmids,
bacteriophages, transposons, insertion elements and genomic islands) (Schmidt & Hensel,
2004).
6.1 Plasmids
Plasmids contribute to genomic plasticity through their transfer capability. They are also
able to mobilize co-resident plasmids and integrate into the chromosome. Plasmids may
harbor antibiotic resistance genes and other genes associated with pathogenicity (Dobrindt
& Hacker, 2001); e.g., Rhodococcus equi harbors a virulence plasmid that codes for surface-
associated proteins (vap genes) that is absent in avirulent strains (Takai et al., 2000).
6.2 Bacteriophages
Bacteriophages are viruses that infect bacteria and which influence genome plasticity
through transduction mechanisms. Functional phages inject DNA from one bacterium into
another one without causing damage to the acceptor organism; the DNA can incorporate
into the acceptor genome leading to adaptive changes. Additionally, prophages (viral DNA
incorporated in the bacterial chromosome) confer protection against lytic infections and they
can harbor virulence genes that may be acquired by the acceptor bacterium and directly
affect its pathogenicity; this has been reported from various species, including Clostridium


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botulinum, Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli and C.
diphtheriae (Brssow et al., 2004).
6.3 Genomic islands
Genomic islands (GEIs) affect genome plasticity because of their mobility and their
capability of carrying a large number of genes as a single block, including operons and
groups of coding genes with related functions. These GEIs can cause dramatic changes that
lead the acceptor bacterium to evolve very rapidly compared to wild-type counterparts.
GEIs are characterized as large DNA regions acquired from other organisms. They vary in
size (10-200 kb), and can harbor sequences derived from phages and/or plasmids, including
integrase genes; GEIs are flanked by tRNA genes or direct repeats, which help produce their
characteristic instability (Hacker & Carniel, 2001). The instability of GEIs is exemplified by
rapid gene acquisition and/or loss and changes in gene composition, as seen in different
strains of Burkholderia pseudomallei (Tumapa et al., 2008). Additionally, GEIs can be
classified into several classes according to gene content. These include Symbiotic Islands,
which are involved in the association of bacterium with Leguminosae hosts (Barcellos et al.,
2007); Resistance Islands, which harbor genes related to antibiotic resistance (Krizova &
Nemec, 2010); Metabolic Islands, which contain genes associated with secondary metabolite
biosynthesis (Tumapa et al., 2008); and Pathogenicity Islands (PAIs), which have a high
concentration of virulence genes. PAIs are associated with pathogenic bacteria and have
been implicated in the reemergence of various pathogens as causes of serious disease
problems (Dobrindt et al., 2000). The first description of a PAI was made in 1990, in vitro
(Hacker et al., 1990),. The identification was based on the observation of a close relation
between deletion of hemolysin and fimbrial adhesin coding regions and non pathogenic
strains of E. coli. This was investigated by gene cloning technique, pulse field
electrophoresis and Southern hybridization. Using these procedures, they showed that the
hemolysin and fimbrial adhesin coding genes are located in the same chromosomal region
in several wild-type strains of E. coli and that they go through deletion events both in vivo
and in vitro (Hacker et al., 1990).
6.4 "Black Holes"
Additionally, it is important to keep in mind that gene deletion is just as important as gene
acquirement in some organisms. One example of this event is the so called "Black Holes" or
deletion events of "antivirulence" genes, i.e. genes whose expression in pathogenic
organisms is incompatible with virulence. The concept of evolution through deletion of
"antivirulence" genes is based on the premise that genes required for adaptation of one
organism in a specific niche may inhibit adaptability in another niche, a potential host, for
example (Maurelli, 2007). In E. coli, loss of cadA, the lysine decarboxylase (LDC) coding
gene, and ompT, which synthesizes an outer membrane protein, may confer virulence
(Suzuki & Sasakawa, 2001). The mechanism of action of cadaverine, produced by
decarboxylation of lysine by LDC, is still unknown. However, there are two hypotheses:
cadaverine inactivates the synthesized toxin, or cadaverine acts directly on the target cell to
protect it. Maurelli et al. (1998) demonstrated that when rabbit mucous cells are pre-treated
with cadaverine and then washed, they are protected from enteroxin effects. Absence of
Omp-T in Shigella strains and enteroinvasive E. coli strains is crucial for maintaining VirG
on the cell surface, a pre-requisite for mobility on mammal cells, including bacterial
dispersion through epithelial cells (Suzuki & Sasakawa, 2001).


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6.5 Software to identify horizontal gene transfer (HGT) events
Gene acquisition and loss through HGT influence bacterial lifestyles and their physiological
versatility (Dobrindt & Hacker, 2001). The increasing number of complete genome
sequences available for analysis has stimulated in silico research in an effort to identify HGT
events. Horizontallyacquired regions can be identified based on observation G+C content
and codon usage patterns, which differ among species and species groups. Sets of genes
acquired by HGT events show deviations in these patterns that reflect the genomic signature
of the donor genome (Langille et al., 2008). Various softwares can be used to identify HGT
events based on base composition patterns (wavelet analysis of G+C content, cumulative GC
profile, Pweb, IVOM, IslandPath and PAIIDA) and codon usage deviation (SIGIHMM
and PAIIDA). However, due to adaptations in codon usage (Karlin et al., 1998), which tend
towards homogenous base composition distributions (Hershberg & Petrov, 2009),
identification of mobile regions based on genomic signature is only possible for regions that
have recently been acquired from phylogenetically distant organisms, i.e. those that have a
discrepant genomic signature when compared to the acceptor genome.
Additionally, identification of HGT events may be aided by concentrating on regions that
are flanked by tRNA genes, which are "hot spots" for transfer elements since they possess 3'
terminal insertion sequences that are recognized by various integrases (Hou, 1999). The
integration of PAIs into these insertion sequences is responsible for their instability, since a
single integrase may cause excision of the entire region. Insertion/deletion events have been
demonstrated in PAIs I and II of E. coli strain 536, which are flanked by selC and leuX tRNA
genes (Blum et al., 1994), and in high pathogenicity islands (HPIs) of several Yersinia
pseudotuberculosis and Y. pestis strains (Lesic et al., 2004), which frequently insert into ASN3
tRNA genes.
However, although efficient in the identification of HGT events, approaches based on
genomic signature and flanking tRNAs are not aimed at classification of GEIs, since they do
not consider the overall gene content of the region. Additionally, horizontally acquired
regions may deviate only in G+C content or codon usage alone, which would be a problem
for the identification process if only one of these features is used to identify the event.
However, there are tools designed to identify a specific class of GEIs, pathogenicity islands,
through a multi-pronged strategy that overcomes such constraints. These tools are named
PredictBias (Pundhir et al., 2008), IslandViewer (Waack et al., 2006) and PIPS (unpublished);
they perform analyses based on genomic signature deviations that are not found in closely-
related organisms and finding of genes coding for virulence factors. Although all of these
programs use similar strategies and are complementary, PIPS deserves special attention
since it surpasses the others in accuracy and is easy to install.
In analysis of C. diphtheriae strain NCTC 13129, PIPS outperformed the other approaches,
identifying 12 out of the 13 PAIs of the reference strain, compared to 10 by IslandViewer
and six by PredictBias. In the identification of PAIs of uropathogenic E. coli strain CFT073,
PIPS had an overall accuracy of 93.9% (unpublished) against 89.5% for IslandViewer and
88.1% for PredictBias.
7. Reverse vaccinology
Reverse Vaccinology (RV) (Rappuoli, 2000) starts from the genomic sequence of a pathogen,
which is an expected coded sequence for all the possible genes expressed during the life
cycle of the pathogen. All open reading frames (ORF's) derived from the genome sequence


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can be evaluated with a computer program in order to determine their aptitude as vaccine
candidates. Special attention is given to exported proteins because they are essential in host-
pathogen interactions. Examples of this interaction can be cited: (i) adherence to host cells,
(ii) invasion of compliant cells, (iii) damage to host tissues, (iv) resistance to environmental
stress by the machinery defense of the cell being infected and finally, (v) mechanisms for
subversion of the host immune response (Sibbald & van Dij, 2009). The word "Reverse" in
RV can be explained by the reverse genetics (RG) technique. Before the dawn of genomics,
there were attempts to discover the genes responsible for each phenotype. With Crick's
central dogma (DNA > RNA > Protein) the research path was reversed. In possession of the
likely gene sequence, several techniques have been developed to identify changes in the
phenotype of an organism derived from sequence changes in genes. The principle of Crick's
dogma is also used by RV; when a gene sequence is found, one can determine whether a
probable protein encoded by this sequence can be an antigen capable of stimulating an
immune response in a host organism.
Long before the creation of the term RV, a number of approaches had been considered to
determine exported proteins in order to move to the next step of the production of a subunit
vaccine (Diaz Romero & Outschoorn, 1994). For example, research on exported proteins was
advanced as an alternative to subunit vaccines based on the polysaccharide capsule of
meningococci. Vaccines produced with such antigens had a low capacity to induce a
satisfactory immune response. This research effort on exported proteins includes almost two
decades of work searching for a vaccine against meningococcal serogroup B, which now
gives good results. This vaccine currently is the best RV alternative for the production of a
subunit vaccine for Neisseria meningitidis serogroup B. Meningitis caused by serogroup B
(Men B) is responsible for approximately half of the worldwide incidence of this disease
(Diaz Romero & Outschoorn, 1994), and this research result for targeted vaccination is
commonly used as a demonstration of the usefulness of RV, because of the excellent results.
Currently, a subunit vaccine against Men B created with antigens targeted by RV is being
tested in phase-2 clinical trials (Bambini & Rappuoli, 2009). The advantages of RV continue
to be attractive, enabling vaccine research for organisms whose cultivation in the laboratory
is difficult or impossible. Reducing the time needed to select target proteins could allow
investigation of different species or strains at the same time, for selecting vaccine candidates
that can elicit adaptive immune responses. To achieve these benefits all we need is to have a
sequenced genome, a personal computer and core software widely available to the scientific
community. These conditions demonstrate another advantage of using RV, the low cost.
What we call core software is a set of tools for identifying well-known motifs, such as, for
example, SignalP, TMHMM, LipoP, and HMMSEARCH. There is still room for innovation
in the use of core software; the choice of software strategies can be directed to the
identification of vaccine candidates specific to an organism, such as in the case of gram-
negative (bilayer) or gram positive (monolayer) bacteria, or also according to heuristics for
selection of vaccine candidates with specific characteristics. For example, membrane or
exported to the extracellular environment (Barinov et al., 2009).
The concept of RV was adapted to fit a new reality of widespread availability of genomic
data (Rinaudo et al., 2009). Instead of researching vaccine targets for a single strain or
subspecies of an organism, we can do it simultaneously in dozens of genomes, exploring
potential joint antigens or those exclusive to multiple genomes (Lapierre & Gogarten, 2009).
The possibility of having a large number of genomes available to implement RV leads to the


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emergence of the concept of pangenomics RV (PGRV) (Bambini & Rappuoli, 2009). PGRV
can also apply the concepts of core, extended, and character genomes. The core genome in
PGRV is composed of exported genes (genes that transcribe exported proteins) that are
common to all strains, genes that could be candidates for a universal vaccine, while the
extended genome consists of genes that are absent in at least one of the strains of the studied
species, while the character genome consists of genes that are specific to a strain (Lapierre &
Gogarten, 2009). From the standpoint of vaccines, the core and character genomes would be
good candidates to develop a vaccine that is suitable for all strains, without losing sight of
the particularities of specific genes in each strain.
8. Immunoinformatics
The immune system has considerable diversity in its components, such as, for example,
immunoglobulin receptors of lymphocytes, or cytokines, with the principle cell types being
B- and T-cells, which have important roles in inflammation, infection and protection (Evans,
2008). Immunoinformatics is very complex and can be characterized as a combinatory
science, since it has a great complexity of regulatory cycles and network type interactions,
which allows the utilization of computational models to resolve problems that can be
converted into biological significant responses (Brusic & Petrovsky, 2003). This leads us to
immunoinformatics, which is the application of informatics techniques to immune system
molecules, with the main objective of helping develop vaccines through the prediction of
immunogenic epitopes (Flower & Doytchinova, 2002).
8.1 Immunological databases
The immunological databases are a source of data used to explore, refine and develop new
tools and algorithms (Salimi et al., 2010). There is a large variety of databases that group
information relevant to the immune system. The Nucleic Acids Research Molecular Biology
Database Collection http://www3.oup.co.uk/nar/database/c/ included 29 immunological
databases in March 2011. The International ImMunoGeneTics information system (IMGT),
the world reference databank for immunogenetics and immunoinformatics, was created by
Marie-Paule Lefranc in 1989 (Lefranc et al., 2009). This databank is specialized in
immunoglobulins or antibodies, T-cell receptors (TCR), MHCs, and others. The IMGT is
constituted of a variety of databanks, including: structure, monoclonal antibody, sequence
and genome databanks. All of these databanks are curated manually and daily by a team
that works fulltime, which helps maintain high-quality annotation and standardization of
the information. Other databases that house information related to epitopes, such as AntiJen
(Toseland et al., 2005) and FIMM (Schonbach et al., 2000), have not been maintained and
their data has migrated to other websites. Among these, the most promising epitope
database seems to be the Immune Epitope Database (IEDB) (Peters et al., 2005), which is a
curated database that has information based on experimental data associated with the target
epitope; consequently, it is hoped that all of the information in the various existing
databanks also migrates to IEDB within the next few years.
8.2 Epitope prediction
The principal goal of immunoinformatics is the development of algorithms that can both
help develop vaccines and analyze the gene products of pathogens, such as viruses and


696
bacteria. This is why it is very important to understand antigen-antibody interactions.
Macallum et al. (1996) made a detailed analysis of 26 antigenantibody complexes; they
found that binding between molecules is very complex, and that there are different
antibodyantigen classes for different types of molecules. A later study of 59 antigen
antibody interactions (Almagro, 2004) found results similar to those of Macallum. These
studies show that tools that can identify molecules and predict their interactions with other
molecules need to be very accurate and sensitive.
8.2.1 B cell epitope prediction
Epitopes of B cells are antigenic regions that are recognized by antibodies of the immune
system, specifically those that interact with B cell receptors. These epitopes can be
continuous or discontinuous (Kumagai & Tsumoto, 2001). Bcell epitopes can be used to
design vaccines and new diagnostic tests (Larsen et al., 2006). As with T cells, there are also
numerous methodologies to model and predict Bcell epitopes. The classic system to predict
Bcell epitopes (Hopp & Woods, 1981) uses propensity scale methods (Parker et al., 1986;
Levitt, 1978). This method attributes a propensity value to each amino acid, based on studies
of the physicalchemical properties. A combination of various scales can improve the
prediction results (Pellequer et al., 1991). This work used hydrophilicity scales (Parker et al.,
1986), as well as secondary structure (Levitt, 1978; Chou & Fasman, 1978) and accessibility
(Emini et al., 1985). The Immune Epitope Database and Analysis Resource, IEDB (Peters et
al., 2005), utilizes parameters such as hydrophilicity, flexibility, accessibility, turns, exposed
surface, polarity and antigenic propensity of polypeptides chains, which have been
correlated with the location of continuous epitopes. All of the prediction calculations are
based on propensity scales. Another methodology that can be used to predict continuous B-
cell epitopes combines hidden Markov model (HMM) and propensity scale methods (Parker
et al., 1986; Levitt, 1978); it is called Bepipred http://www.cbs.dtu.dk/services/BepiPred/
(Larsen et al., 2006). This methodology has given increased prediction accuracy. Prediction
of discontinuous Bcell epitopes has also improved, due to an increase in the number of
threedimensional (3D) structures of antibodyantigen complexes available in PDB and in
IMGT/3Dstructure-DB (Kaas et al., 2004) and in the Epitome database (Schlessinger et al.,
2006).
8.2.2 T cell epitope prediction
There are two classes of T cells: (1) CD8+ T cytotoxic (Tc) cells, which produce cytotoxins
responsible for cell lysis, recognize peptides presented by class I MHCs and (2) CD4+ T
helper (Th) cells, which recognize proteins associated with MHC class II. Interferon (IFN
) and tumor necrosis factor (TNF) are produced by Th1 cells. Th2 cells produce
interleukin 4 (IL4), IL5, IL10 and IL13. Eptitopes that bind to MHC de class I generally
are 810 amino acids long, with a mean of nine amino acids (Reche et al.., 2002), while
epitopes that bind to MHC class II are 1317 amino acids long (Sercarz & Maverakis, 2003;
Chicz et al., 1992). There are various online tools for predicting Tcell epitopes on the basis
of MHC class I and class II binding. Prediction of MHC binding is based on motifs
associated with epitopes or binders for specific alleles. SYFPEITHI is a tool that is widely
used for prediction of Tcell epitopes and MHC binding; however, these predictions have
been found to be of low quality (Ruppert et al., 1993). More sophisticated tools that use
quantitative matrixes, artificial neural network decision trees, hidden Markov models


697
(HMM), support vector machines (SVM), homology modeling, protein threading and
docking techniques have been developed. The NetMHC 3.2 server
http://www.cbs.dtu.dk/services/NetMHC/ predicts binding of peptides to a series of
different HLA alleles using artificial neural networks (ANNs) and weight matrixes. All of
the previous versions are available online, for comparison and reference. ANNs were
trained with 57 different human MHCs (HLA), representing all of the 12 HLA alleles,
supertypes A and B (Lund et al., 2004). Also predictions are available for 22 animal alleles
(monkey and rat). ANN prediction values are given in nM IC50 values. Weight prediction
matrixes use an aptitude score, with a high aptitude score indicating strong binding.
Predictions can be made for sizes from 8 to 11 for all of the alleles using an ANNs algorithm
trained with 9mer peptides. Probably because of the limited quantity of 10mer data
available, this method has better prediction value when an ANNs algorithm is trained with
10mer data. However, one should be careful with 8mer predictions, since some alleles do
not link to 8mer to a significant degree. Binding peptides are indicated at output as strongly
binding (SB) and weakly binding (WB). The allele for each HLA supertype is indicated in
the selection window for HLA alleles (Lundegaard et al., 2008).
The NetMHCII 2.2 server http://www.cbs.dtu.dk/services/NetMHCII/ predicts
peptides that bind to MHC classe II alleles HLADR, HLADQ, HLADP and mouse alleles,
using ANNs. Predictions can be obtained for the 14 HLADR alleles, including the nine
HLADR, six HLADQ, and six HLADP supertypes and two H2 class II alleles in mice.
The prediction values are given in nM IC50 values, and in %Rank for a random set of
1,000,000 natural peptides. Strongly and weakly binding peptides are indicated in the
output file (Nielsen et al., 2007).
Without a doubt, there is a great variety of predictors, which when they are combined can
be quite precise in the prediction of Tcell epitopes; however, this is only possible when
wellcharacterized alleles are available, which is true for some alleles that have been
predicted as MHC class I alleles, but much less so for those predicted as MHC class II. This
is even more of a problem in the prediction of B cell proteins, for which it is often necessary
to have prior knowledge of the structure and sequence of the protein. Nevertheless, it is
known that no method can go further than the data used to train it, and only through
extensive compilation and by obtaining high quality data, will it be possible to create
excellent models that will can be generally applied (Flower & Doytchinova, 2002).
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Biotechnol.
Part 10
Drug Design

31
Designing of Anti-Cancer Drug Targeted to
Bcl-2 Associated Athanogene (BAG1) Protein
Amit Kumar, Kriti Verma and Amita Sinha
Department of Bioinformatics and MolecularBiology,
Bioaxis DNA Research Centre, Hyderabad,
India
1. Introduction
Cancer is a disease of uncontrolled cell growth in tissues. This growth may lead to
metastasis, which is the invasion of adjacent tissue and infiltration beyond the site of
initiation. Cancer is initiated by activation of oncogenes or inactivation of tumor suppressor
genes. Nearly 10-30% of all adenocarcinomas are due to the mutations in the K-ras proto-
oncogene. [1] Function and regulation of Bcl-2 proteins depends upon their interaction with
other non-family member proteins, including NIP1, NIP2, NIP3, p53 BP2, Raf-1, CED-4,
calcineurin, R-Ras and Bag-1 to form homo and hetero dimmers.[21] Bag1 belongs to the Bcl-
2 associated athanogene (BAG) family of multifunctional proteins. This widely expressed
protein interacts with a number of signalling molecules (including Bcl2, HGF receptor and
Raf1) as it regulates signalling molecules in pathways involving cell survival, growth and
differentiation. [13] Bcl2 associated athanogene (BAG1) protein is involved in regulation of
the Ras/Raf signal transduction pathway. Of particular relevance to tumour cells, BAG-1
interacts with the anti-apoptotic BCL-2 protein, various nuclear hormone receptors the 70
kDa heat shock proteins, Hsc70 and Hsp70; and serine/threonine kinase. Raf-1 which plays
an important role in MAPK pathway.[2][3][4] Recent studies have shown that BAG-1
expression is frequently altered in malignant cells, and BAG-1 expression may have clinical
value as a prognostic or predictive marker for various cancer types including breast cancer,
prostate cancer and lung cancer.[6][7][8] (Fig 1) Interaction with chaperones may account for
many of the pleiotropic effects associated with BAG-1 over expression. The finding that
BAG-1 can independently associate with Raf-1 or Bcl-2 provides at least two mechanisms by
which BAG-1 promotes cell survival. [20]
Bcl2-associated athanogene (BAG) family proteins participate in a wide variety of cellular
processes to regulate growth control pathways, including cell survival (stress response),
proliferation, migration, signalling and apoptosis (Fig 2).[2][5][18] This family of co-
chaperones functionally regulates signal transduction proteins Raf/MEK/ERK and
transcription factors important for cell stress responses, apoptosis, proliferation, cell
migration and hormone action. In response to stress, they bind to heat shock proteins
HSP70/HSC70 coordinating cell growth signals, by down-regulating the activity of
serine/threonine kinase, Raf-1, which plays an important role in MAPK pathway. [5][9] The
proteins show anti-apoptotic activity and increase the anti-cell death function of BCL-2
induced by various stimuli. Over expression of BAG-1 suppresses activation of caspases and


708
apoptosis induced by a very broad range of agents in different cell types, for example
chemotherapeutic agents, radiation and growth factor withdrawal. Therefore, in addition to
contributing to reduced cell death in cancer development, BAG-1 may also contribute to
resistance to important therapeutic modalities.

Fig. 1. BAG1 expression in normal and diseased human tissues.
BAG-1 proteins are expressed as multiple isoforms generated by alternate translation
initiation from a single mRNA. Translation of the major human BAG-1 isoform, BAG-1S,
initiates at an internal AUG codon, whereas of the larger BAG-1L (p50) and BAG-1M
proteins translation begins upstream at CUG and AUG codons, respectively.[6][9] Hence,
the proteins share a common C-terminus. However, the larger isoforms have additional N-
terminal sequences. Various domains have been identified within BAG-1 proteins. [14] A
potential nuclear localisation signal (NLS) within the unique N-terminal domain of BAG-1L
has been identified. However, BAG-1S and BAG-1M lack this sequence. BAG-1S is largely
located in the cytoplasm in contrast to BAG-1M which partitions between the nucleus and
cytoplasm. [8]
At the carboxy terminal of all BAG-1 isoforms there is a conserved region of about 110
amino-acids, named as the BAG domain, which binds and regulates Hsp70/Hsc70
molecular chaperones. [8][23] BAG domains are present in Bcl-2-associated athanogene 1
and silencer of death domains.
The crystal structure of the BAG domain revealed that it consists of three anti-parallels
helices. In the BAG domain the first and the second -helices interact with the
serine/threonine kinase Raf-1 and the second and third -helices interact with the ATP-
binding pocket of Hsc70/Hsp70. Therefore, Raf-1 and Hsp70/Hsc70 have partially
overlapping sites and their binding is competitive. [2] [8]
BAG-1 promotes cell growth by binding to and stimulating Raf-1 activity. The binding of
Hsp70 to BAG-1 diminishes Raf-1 signalling and inhibits subsequent events, such as DNA
synthesis, as well as arrests cell cycle. When cellular levels of Hsp70 are elevated during
stress, or in cells conditionally over expressing Hsp70, Bag1-Raf-1 is displaced by Bag1-
Hsp70, and DNA synthesis is arrested.[5][10] Thus, BAG-1 has been suggested to function as

Designing of Anti-Cancer Drug Targeted to Bcl-2 Associated Athanogene (BAG1) Protein

709
a molecular switch that controls cells to proliferate in normal conditions but become
quiescent under a stressful environment.[16]
The C-terminus of the BAG domain is also a site of interaction with Bcl-2 which provides a
supra-additive anti-apoptotic effect. The BAG-1 protein shares no significant homology with
Bcl-2 or other Bcl-2 family proteins, which can form homo- and heterodimers. [11]
All BAG-1 isoforms also contain an ubiquitin-like domain (ULD), similar to ubiquitin and
ubiquitin-like proteins that appears to be essential for at least some biological effects[8][7][15].
Although the precise function of the ULD in BAG-1 is unknown, BAG-1 isoforms are very
stable proteins suggesting that they are not generally targets for degradation by the
ubiquitin/proteasome system and are not covalently attached to other proteins.
2. Role of BAG1 and Bcl2 in apoptosis
Bcl-2 is an anti-apoptotic protein located mainly on the outer membrane of mitochondria. It
has been found that over-expression of Bcl-2 inhibits

cells from undergoing apoptosis in
response to a various stimuli. [12] The members of the Bcl-2 family share one or more of the
four characteristic domains of homology entitled the Bcl-2 homology (BH) domains (named
BH1, BH2, BH3 and BH4).[11][13] The BH domains are known to be crucial for its function,
as deletion of these domains via molecular cloning affects survival/apoptosis rates. The
anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Bcl-xL, conserve all four BH domains. Bcl-2
interacts with pro-apoptotic proteins BAX and BAK. The hydrophobic unit of Bcl-2 forms a
heterodimer with the amphipathic unit of BAX and BAK. This heterodimer formation
inhibits release of cytochrome c from the mitochondria and prevents activation of caspases.
The protein encoded by BAG1 gene binds to BCL2 and is referred to as BCL2-associated
athanogene. It enhances the anti-apoptotic effects of BCL2. [12][13]

Fig. 2. Interaction of BAG1 protein with other proteins and cellular components. [8]


710
3. Role of Raf-1/ MAPK pathway in cancer
The pathways regulated by BAG-1 play key roles in the development and progression of
cancer and determining response to therapy. The extracellular signal-related kinase (ERK),
among the MAPK pathways, plays a key role in promotion of cellular proliferation,
survival, and metastasis, this pathway directly affects the initiation and progression of
human tumors. This pathway has been found to be activated in numerous cancer types
without obvious genetic mutations. Cell lines derived from various organs such as pancreas,
colon, lung, ovary and kidney have been reported to show a high degree of MAP kinase
activation as observed in 50 tumor cell lines. However, it ihas been found that the
constitutive activation of MAP kinases in tumor cells is not due to the disorder of MAP
kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling
molecules upstream of Ras.[3]
The Ras/Raf/MEK/ERK pathway also interacts with the p53 pathway thereby regulating
the activity and subcellular localization of BCl2 family proteins (Bim, Bak, Bax, Puma and
Noxa). Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of
apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages.[3][4]
4. Methodology used in present study
The strategy used in this project is to target the first alpha helix of the BAG domain. Binding
of a ligand to the first alpha helix provides two simultaneous scenarios i.e. firstly it blocks
the site for Raf1 binding and thus blocks the MAPK pathway. Secondly, it makes the second
and third alpha helix available for Hsp70 binding. Binding of Hsp70 to BAG1 protein
renders the heat shock protein inactive as BAG1 has been found to have inhibitory effect on
Hsp proteins. This shall produce pseudo stress conditions and attenuate DNA synthesis and
cellular proliferation.
Thus, the aim of the present study is to design a drug, targeted to the first alpha helix of
BAG Domain of BAG1 protein that binds to the competitive binding site of Raf1 and Hsp70
thereby blocking the binding site of Raf1, making it available for Hsp70 binding and hence
suppressing its anti-apoptotic activity. The therapeutic goal is to arrest further tumor
progression and trigger tumor-selective cell death by disrupting the balance between pro-
apoptotic proteins and anti-apoptotic proteins (Fig 3).
5. Important tools and databases
NCBI is a primary database majorly used for sequence retrieval and similarity based
searches. We used NCBI for our sequence retrieval of query protein sequence. BLAST is the
most widely used sequence similarity search programme. It finds regions of local similarity
between sequences. In this study protein blast has been extensively used. PubMed database
was primarily used for literature search including journals, abstracts, full text articles and
other sources related to the research. PDB is repository for the 3-D structural data of large
biological molecules, such as proteins and nucleic acids which is obtained by X-ray
crystallography and NMR spectroscopy. PDB was used to retrieve the 3D structure of the
protein. Biology Workbench is a web based tool integrated with access to a wide variety of
analysis and modeling tools. This tool has been used for phylogenetic analysis of the Bag1
protein sequences.


711
Clustalw is a multiple sequence alignment program that calculates the best match for the
selected DNA or protein sequences and then lines them up so that the identities, similarities
and the differences can be seen. Boxshade works by global alignment of all sequence.
Conserved and similar residues are emphasized by various degrees of shading. KEGG
database was used for pathway analysis for this Bag1 protein. Genecards has been used in
this work to get various expression and sequence related information pertaining to proteins
of the Bag1 protein. In this work all the above programs have been used to obtain the
peptide recognition pattern for the interpretation of results.

Fig. 3. Flowchart depicting the drug targeted strategy.


712
6. Structure analysis tools
ProtParam is a tool which allows the computation of various physical and chemical
parameters for a protein sequence. The computed parameters include the molecular weight,
theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated
half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY).
The HNN method was used for secondary structure prediction for Bag1 protein.
CPHmodels is a web server predicting protein 3D structure

by use of single template
homology modeling. The CPHmodels server predicts protein structure from amino acid
sequence with respect to distance constraints. CPHmodels is a collection of methods and
databases consisting of the following tools: CPHmodels was used for tertiary structure
prediction of Bag1 protein.
The 3d structure obtained as a pdb file format was viewed using RasMol.
PRODOM and PROSITE is a comprehensive database of protein domain and families.
PROSITE offers tools for protein sequence analysis and motif detection.
STRING is a database of known and predicted protein interactions.
CASTP (Computed Atlas of Surface Topography of Proteins) is a server that provides
identification and measurements of surface accessible pockets as well as interior inaccessible
cavities for proteins and other molecules.
7. Drug discovery tools
Drugbank, Pubchem, therapeutic target database (TTD), Tocris are the various databases
which were searched for potential drug candidates.
ArgusLab was used as a molecular modelling program to optimize the target receptor
protein and design a drug targeted to the BAG1 protein. The quantum mechanical
calculations were performed using the Argus compute server.
HEX5.1 is an interactive protein docking and molecular superposition program. HEX5.1 was
used for docking of the selected drug candidate with the BAG1 target protein.
BAG1 isoform 1-L sequence was retrieved from NCBI. The protein BAG1-L is 345 amino
acids in length. Sequence analysis by BLASTp shows that Bag1-L protein showed maximum
identities with Bos Taurus (83%) followed by Mus musculus (80%). BLAST results of BAG1
protein compared with other model organisms is shown in Table 1.
Evolutionary relationship of BAG1 protein among various species was obtained in the form
of a dendrogram (Fig.4) The query protein was seen to be most closely related to Mus
musculus and showed a distinct evolutionary relationship with Suberites domuncula. The
highly conserved regions in the amino acid sequence of BAG1 in protein among various
model organisms were analysed using Boxshade (Fig. 5). The amino acids Glycine and
Glutamine were found to be the maximum conserved regions among all the species
analysed.
The structural analysis of BAG1-L protein was done by using ProtParam, HNN & CPH.
Since the GRAVY value is negative (-0.905) it can be inferred from the results that the
protein is a hydrophobic molecule present at the cell surface. It is an unstable protein. It
consists of 40.00% alpha helices and no beta bridges or turns are present. (Fig. 6) Using CPH
model, the 3D structure of the protein was retrieved in the form of a PDB ID. The PDB ID


713
with the maximum score i.e. 1HX1 was chosen and viewed in Rasmol. (Fig. 7) The PDB ID
1HX1 shows the 3D structure for two molecules Hsp70 and Bag domain as Chain A (400aa)
and Chain B (114aa) respectively. The Chain B i.e. Bag domain (receptor) was isolated and
its energy was optimized to 3734.78 au using ArgusLab. The molecule converged at 298.92
kcal\mol.
CASTP was used to find the active pocket in the receptor protein. The amino acid Lysine in
Chain B (receptor molecule) at position 172 is selected as the target residue for Arguslab
docking as it is the most hydrophilic residue in the active pocket.
A number of small molecules that bind to the target were searched by screening libraries of
potential drug compounds. The toxic effects and pharmacodynamics of the compounds was
tested by ADME/Tox. The compound Carmustine was chosen out of the drug library as it
followed the Lipinskis rule of five and had the best combination of required properties for a
potential drug candidate. (Table 2)
The molecule Carmustine was designed in ArgusLab and geometry optimization of the
drug got converged at energy of 22.2 Kcal\mol. Total energy of the compound converged at
-101.413 au.
The drug was docked to the target residue using Arguslab and Hex5.1. In Hex, the drug
docked to the receptor with an Emax value (Energy) of -94.68 kcal\mol and Emin value of -
166.49 kcal\mol. (Fig. 9) In Arguslab, the drug docked to the target receptor with energy of -
5.51 kcal\mol.

Fig. 4. Dendogram depicting the phylogenetic relationship of Bag1 (Query) protein in Homo
sapiens with Bag1 protein of other model organisms (humans).


714

Fig. 5. Boxshade showing some of the highly conserved (green) and similar (cyan) pattern
for Bag1 protein in different model organisms.

Fig. 6. The secondary structure analysis result of Bag1 by HNN tool. The alpha helices are
shaded blue, beta sheets are shaded red.


715

Fig. 7. Visualization result of Bag1protein using the tool RASMOL showing the 3D structure
details.

Fig. 8. STRING analysis of BAG1 protein showing its interaction with other proteins in the
human. The number of lines represents the strength of interaction.


716

Fig. 9. Docking analysis result of Bag1 protein with the drug Carmustine in Hex5.1.


717

Model
Organism
Name
BLAST
Results
Homo sapiens
GENE ID: 573 BAG1 | BCL2-associated athanogene [Homo sapiens]
(Over 10 PubMed links)
Score = 724 bits (1671), Expect = 0.0, Method: Compositional matrix
adjust.
Identities = 345/345 (100%), Positives = 345/345 (100%), Gaps = 0/345
(0%)
Bos taurus
GENE ID: 613855 BAG1 | BCL2-associated athanogene [Bos taurus]
Score = 413 bits (950), Expect = 1e-115, Method: Compositional matrix
adjust.
(0%)
Mus musculus
GENE ID: 12017 Bag1 | BCL2-associated athanogene 1 [Mus musculus]
adjust.
(0%)
Gallus gallus
GENE ID: 420967 BAG1 | BCL2-associated athanogene [Gallus gallus]
(10 or fewer PubMed links)
adjust.
(0%)
Rattus
norvegicus
GENE ID: 297994 Bag1 | BCL2-associated athanogene [Rattus
norvegicus]
adjust.
(3%)
Dictyostelium
discoideum
GENE ID: 8616246 sonA | UAS domain-containing protein
[Dictyostelium discoideum AX4] (10 or fewer PubMed links)
Score = 34.5 bits (102), Expect = 0.053, Method: Compositional matrix
adjust.
Identities = 17/49 (34%), Positives = 29/49 (59%), Gaps = 1/49 (2%)
Caenorhabditis
elegans
GENE ID: 172373 bag-1 | BAG1 (human) homolog [Caenorhabditis
elegans]
Score = 51.5 bits (160), Expect = 8e-07, Method: Compositional matrix
adjust.


718
Model
Organism
Name
BLAST
Results
Suberites
domuncula
emb|CAJ65915.1| BAG family molecular chaperone regulator 1
[Suberites domuncula]
Length=258
adjust.
Brugia malayi
GENE ID: 6105907 Bm1_55120 | BAG domain containing protein [Brugia
malayi]
adjust.

Table 1. Showing the result of query sequence BLAST with different model organisms.

Table 2. List of various potential drug candidates for binding with Bag domain of Bag 1
protein.


719
The BAG proteins having anti-apoptotic activity promotes cell growth by binding to and
stimulating Raf-1 activity. BAG-1 binds to the serine/threonine kinase Raf-1 or
Hsc70/Hsp70 in a mutually exclusive interaction. The binding of Hsp70 to BAG-1
diminishes Raf-1 signalling and inhibits subsequent events, such as DNA synthesis, as well
as arrests the cell cycle. Hence Bag1 plays an important role in the progression of cancers
when over expressed. The 345 amino acid long protein sequence of BAG1-L was obtained
from NCBI and a BLASTp was performed to analyze its evolutionary relationships with
other counterparts in various model organisms. This was confirmed by the phylogenetic
analysis done using SDSC workbench. The dendrogram presents that Bag1 had close
evolutionary relationship with Mus musculus.
From the primary structure analysis it was concluded that BAG1 protein is a surface protein
which is hydrophilic in nature. Its secondary structure analysis confirmed that it contains
more alpha helices and no beta sheets. Its 3D structure was obtained in the form of PDB id
and viewed in Rasmol. The chain B of PDB structure represents the BAG domain. Various
confirmatory tools were used for validation of the results. The geometry and energy of the
BAG domain was optimized in Arguslab. Using Castp, the active pocket in the BAG
domain was identified and the most hydrophilic residue in the first alpha helix of the BAG
domain i.e. LYS at position 242 of BAG1-L protein sequence obtained from NCBI (or
position 172 in the Chain B of pdb id 1HX1) was selected as the target receptor.
A drug library was maintained of possible lead compounds that follow the Lipinski rule of
five and their toxicity and disposition was checked using ADME/TOX. These candidate
drugs were docked to the target receptor. The drug CARMUSTINE showed the best docking
result with docking Energy of -5.51kcal/mol. As the docking (Fig 9) was successful in both
HEX5.1 and Arguslab it can be concluded that Carmustine can be a potential drug for BAG1
binding and arresting tumor progression. Further analysis must be performed on this drug
for use in treatment of cancer.
9. Acknowledgements
The work was done by worthwhile efforts of the staff and the research associates of the
Department of Molecular Biology Bioaxis DNA Research Centre, Hyderabad, India. In
addition, the authors would like to thank all the technical staff of instrumental section in
developing and maintaining the various databases and tools mentioned in this article.
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is a length random sequence generated

be an observed sequence of length over /. Then the number N(/; X

(a) NFA for A

(b) NFA for C

(c) NFA for G

(d) NFA for T

the set of all nite sequences over /. Any subset / /

) the set of all possible languages over /. We denote

): For all / T(/

) dened from singleton

GGATG is a regular language, AG, AAGG, AAAGGG, . . . is not a regular language.

is accepted by this NFA

): / regular it exists a NFA whose language is

and the three

we build the NFA (/, Q, , T, ) with Q = 0, 1, . . . , , = 0,

G(G[C)G. Then we directly apply

is roughly equal to = 25 (we will see later on how

/ from which we build a DFA (/, Q, q

/ and with the property that for

) which could be slightly smaller than Q.

G(G[C)G. The order 1 past of each

. We then connect this

, N). Finally, we sum up the

the number of matching position in X

atoms with a r.m.s.d. of 0.69 . Arrows indicate flexible loops.

be the set of the metabolites and M

be the set of the reactions in a special case

), where each node

and each arc represents a reaction k M

, we redene the stoichiometric matrix S

J be the set of metabolites that will

. By using the stoichiometric matrix S

, Circularity: Shape of spot defined as

Grady, A. (2003). Tissue microarray: an overview. Current Diagnostic

measure (for non-negative real values of ).

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